Teaching molecular genetics: chapter 4—positional cloning of genetic disorders

Positional cloning is the approach of choice for the identification of genetic mutations underlying the pathological development of diseases with simple Mendelian inheritance. It consists of different consecutive steps, starting with recruitment of patients and DNA collection, that are critical to the overall process. A genetic analysis of the enrolled patients and their families is performed, based on genetic recombination frequencies generated by meiotic cross-overs and on genome-wide molecular studies, to define a critical DNA region of interest. This analysis culminates in a statistical estimate of the probability that disease features may segregate in the families independently or in association with specific molecular markers located in known regions. In this latter case, a marker can be defined as being linked to the disease manifestations. The genetic markers define an interval that is a function of their recombination frequencies with the disease, in which the disease gene is localised. The identification and characterisation of chromosome abnormalities as translocations, deletions and duplications by classical cytogenetic methods or by the newly developed microarray-based comparative genomic hybridisation (array CGH) technique may define extensions and borders of the genomic regions involved. The step following the definition of a critical genomic region is the identification of candidate genes that is based on the analysis of available databases from genome browsers. Positional cloning culminates in the identification of the causative gene mutation, and the definition of its functional role in the pathogenesis of the disorder, by the use of cell-based or animal-based experiments. More often, positional cloning ends with the generation of mice with homologous mutations reproducing the human clinical phenotype. Altogether, positional cloning has represented a fundamental step in the research on genetic renal disorders, leading to the definition of several disease mechanisms and allowing a proper diagnostic approach to many conditions

Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas

Microsatellite instability has been proposed as an alternative pathway of colorectal carcinogenesis. The aim of this study was to evaluate the interest of immunohistochemistry as a new tool for highlighting mismatch repair deficiency and to compare the results with a PCR-based microsatellite assay. A total of 100 sporadic proximal colon adenocarcinomas were analysed. The expression of hMLH1, hMSH2 and hMSH6 proteins evaluated by immunohistochemistry was altered in 39% of the cancers, whereas microsatellite instability assessed by PCR was detected in 43%. There was discordance between the two methods in eight cases. After further analyses performed on other tumoural areas for these eight cases, total concordance between the two techniques was observed (Kappa=100%). Our results demonstrate that immunohistochemistry may be as efficient as microsatellite amplification in the detection of unstable phenotype provided that at least two samples of each carcinoma are screened, because of intratumoural heterogeneity

Systematic generation of in vivo G protein-coupled receptor mutants in the rat

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies

COVER: Een programma dat dekkingspatronen van radioplaatsbepalingssystemen tekent

Er is door mij een software pakket ontwikkeld, dat dekkingspatronen van radioplaatsbepalingssystemen tekent. Deze patronen zijn in feite lijnen waarop met gelijke standaardafwijking de positie bepaald kan worden. Een logische eis hierbij is, dat de zenders ontvangen moeten kunnen worden. De dekkingspatronen zijn dan ook een combinatie van lijnen van gelijke precisie en lijnen, die de maximale reikwijdte van de zenders markeren.Civil Engineering and Geoscience

Pathogenic sequence for dissecting aneurysm formation in a hypomorphic polycystic kidney disease 1 mouse model

OBJECTIVE - Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1 mouse model with dissecting aneurysms in the aorta. METHODS AND RESULTS - Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding. CONCLUSIONS - The Pkd1 mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies. © 2007 American Heart Association, Inc. Chemicals / CAS: calcium, 7440-70-2; collagen, 9007-34-5; elastin, 9007-58-3; fibronectin, 86088-83-7; gelatinase A, 146480-35-5; gelatinase B, 146480-36-6; osteopontin, 106441-73-0; perlecan, 143972-95-6; versican, 126968-45-4; polycystic kidney disease 1 protein; RNA, Messenger; TRPP Cation Channel