Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool. OBJECTIVE: To present a strategy for mutation detection in families clinically diagnosed with HHT. METHODS: An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT). RESULTS: Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%). CONCLUSIONS: For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.

Neonatal neuronal overexpression of glycogen synthase kinase-3 beta reduces brain size in transgenic mice.

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.

Coexpression of human cdk5 and its activator p35 with human protein tau in neurons in brain of triple transgenic mice.

The potential contribution of cyclin-dependent protein kinase 5 (cdk5) to hyperphosphorylate protein tau, as claimed in Alzheimer's disease, was investigated in vivo. We generated single, double, and triple transgenic mice that coexpress human cdk5 and its activator p35 as well as human protein tau in cerebral neurons. Whereas expression and increased cdk5-kinase activity was obtained, as measured in vitro and demonstrated in vivo, neither murine nor human protein tau was appreciably phosphorylated in the brain of double and triple transgenic mice. These mice behaved and reproduced normally. Silver impregnation and immunohistochemistry of brain sections demonstrated that neurofilament proteins became redistributed in apical dendrites of cortical neurons. This suggested a cytoskeletal effect, but no other relevant brain pathology became apparent. These observations indicate that cdk5/p35 is not a major protein tau kinase and that cdk5/p35 did not cause neurodegeneration in mouse brain, as opposed to cdk5/p25.

Glycogen synthase kinase-3beta phosphorylates protein tau and rescues the axonopathy in the central nervous system of human four-repeat tau transgenic mice.

Protein tau filaments in brain of patients suffering from Alzheimer's disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3beta and GSK-3beta x human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol((R)))-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3beta. Co-expression of GSK-3beta reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153-2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed.

Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.

Mutations in the human tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some mutations, including mutations in intron 10, induce increased levels of the functionally normal four-repeat tau protein isoform, leading to neurodegeneration. We generated transgenic mice that overexpress the four-repeat human tau protein isoform specifically in neurons. The transgenic mice developed axonal degeneration in brain and spinal cord. In the model, axonal dilations with accumulation of neurofilaments, mitochondria, and vesicles were documented. The axonopathy and the accompanying dysfunctional sensorimotor capacities were transgene-dosage related. These findings proved that merely increasing the concentration of the four-repeat tau protein isoform is sufficient to injure neurons in the central nervous system, without formation of intraneuronal neurofibrillary tangles. Evidence for astrogliosis and ubiquitination of accumulated proteins in the dilated part of the axon supported this conclusion. This transgenic model, overexpressing the longest isoform of human tau protein, recapitulates features of known neurodegenerative diseases, including Alzheimer's disease and other tauopathies. The model makes it possible to study the interaction with additional factors, to be incorporated genetically, or with other biological triggers that are implicated in neurodegeneration.