Accurate Prediction of the Functional Significance of Single Nucleotide Polymorphisms and Mutations in the ABCA1 Gene

The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r (2) = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease

The Influence of Huntingtin Protein Size on Nuclear Localization and Cellular Toxicity

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH2-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease

Low Levels of Human HIP14 Are Sufficient to Rescue Neuropathological, Behavioural, and Enzymatic Defects Due to Loss of Murine HIP14 in Hip14−/− Mice

Huntingtin Interacting Protein 14 (HIP14) is a palmitoyl acyl transferase (PAT) that was first identified due to altered interaction with mutant huntingtin, the protein responsible for Huntington Disease (HD). HIP14 palmitoylates a specific set of neuronal substrates critical at the synapse, and downregulation of HIP14 by siRNA in vitro results in increased cell death in neurons. We previously reported that mice lacking murine Hip14 (Hip14−/−) share features of HD. In the current study, we have generated human HIP14 BAC transgenic mice and crossed them to the Hip14−/− model in order to confirm that the defects seen in Hip14−/− mice are in fact due to loss of Hip14. In addition, we sought to determine whether human HIP14 can provide functional compensation for loss of murine Hip14. We demonstrate that despite a relative low level of expression, as assessed via Western blot, BAC-derived human HIP14 compensates for deficits in neuropathology, behavior, and PAT enzyme function seen in the Hip14−/− model. Our findings yield important insights into HIP14 function in vivo

Haploinsufficiency of CYP8B1 associates with increased insulin sensitivity in humans

10.1172/JCI152961The Journal of clinical investigation13221e152961

REGULATION OF HUNTINGTIN PALITOYLATION AND ITS ROLE IN HUNTINGTON DISEASE

Huntington Disease(HD) is an inherited and ultimately fatal neurodegenerative disease demonstrating both neurological and psychiatric symptoms. The protein huntingtin (htt) undergoes many post-translational modifications, such as phosphorylation, palmitoylation, and proteolysis. Palmitoylation, the process by which a 16-carbon fatty acid forms a thioester bond with cysteine residues, is a reversible modification known to influence protein trafficking and function. Huntingtin Interacting Protein 14 (HIP14) was identified as a major palmitoyl acyl transferase (PAT) that interacts robustly with wild-type htt, but has significantly reduced interaction with mutant polyglutamine expanded htt. HIP14is a major PAT for htt and palmitoylation of mutant htt by HIP14 is significantly reduced. Down regulation of HIP14 by siRNA in vitro results in increased cell death in neurons, whereas co-transfection with htt and HIP14 results in enhanced palmitoylation and reduction in number of inclusions. Our laboratory has developed a HIP14 knockout mouse (HIP14-/-), which demonstrates many HD-like features similar to those seen in the YAC128 transgenic mouse model of HD. Notably, these mice demonstrate a much earlier and more severe phenotype as compared to the YAC128 mice, suggesting a critical role for HIP14 in HD pathogenesis. The overarching goal of these studies is to explore the role of HIP14 and palmitoylation in the pathogenesis of HD using in vitro, in vivo, and ex vivo methods. A human Bacterial Artificial Chromosome (BAC) containing HIP14 was identified, prepared, and submitted for microinjection, and we have now generated HIP14-overexpressing transgenic mice. These mice are currently undergoing preliminary analyses, and a subset of founders will be selected to undergo further characterization. These human HIP14 BAC transgenic mice will represent the first mammalian model of PAT overexpression, and will significantly further our understanding of PAT activity in vivo. The YAC transgenic model for HD, developed previously in our laboratory, recapitulates many aspects of HD as seen in humans. After preliminary characterization of the human HIP14 BAC transgenic mice, these mice will be crossed to the YAC model of HD. We anticipate that overexpression of HIP14 in vivo will delay and/or ameliorate the features of HD observed in the YAC128 mouse, providing validation of this pathway as a potential therapeutic target for HD. F.B.J.Y. is supported by a Canadian Institutes of Health Research Walter and Jessie Boyd & Charles Scriver and Child & Family Research Institute –UBC MD/PhDStudentship Award. She also receives funding from the Michael Smith Foundation for Health Research as a Junior Trainee

Efflux and atherosclerosis - The clinical and biochemical impact of variations in the ABCA1 gene

Approximately 50 mutations and many single nucleotide polymorphisms have been described in the ABCA1 gene, with mutations leading to Tangier disease and familial hypoalphalipoproteinemia. Homozygotes and heterozygotes for mutations in ABCA1 display a wide range of phenotypes. Identification of ABCA1 as the molecular defect in these diseases has allowed for ascertainment based on genetic status and determination of genotype-phenotype correlations and has permitted us to identify mutations conferring a range of severity of cellular, biochemical, and clinical phenotypes. In this study we review how genetic variation at the ABCA1 locus affects its role in the maintenance of lipid homeostasis and the natural progression of atherosclerosi