Copy number variation and Huntington’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the expansion of an unstable triplet repeat within the huntingtin gene. The length of this repeat is inversely correlated with the age of onset (AO) of the disease, which ranges from 1 to 80 years of age. The length of this repeat explains 50-70% of the variance of AO with the remaining variation attributable to environmental and other genetic factors. Copy number variation (CNV) is a structural variation of the human genome wherein a genomic sequence is duplicated or deleted compared to a reference genome. As CNVs have the potential to affect gene expression either directly by dosage effects or indirectly by affecting gene product interactions and pleiotropic effects, they are found to be associated with phenotypic variance, disease susceptibility and Mendelian disorders. The aim of the study was to investigate CNVs as candidate genetic modifiers of the AO in HD. Specifically we investigated CNV of the human β-defensin region (including DEFB4) and CNV involving SLC2A3, with potential impacts, respectively, in the neuroinflammatory response and in neuronal glucose uptake. CNVs were analysed within a large HD sample cohort (provided by EHDN) using the paralogue ratio test (PRT) to test their potential impact on a variance of the AO in HD. In 490 HD individuals analysed the frequency distribution of β-defensin copy number was shown to be equivalent to the general European population. Furthermore, no significant association was shown between β-defensin CNV and a variance of the AO in HD. 987 HD patients were genotyped for SLC2A3 CNV and a modest but significant association with a variance of AO in HD was found (p value = 0.028). Individuals with three copies showed a delay in the AO of up to nearly 6 years compared to individuals with one or two copies. In order to test if SLC2A3 CNV affects gene expression 15 cell lines from patients with different SLC2A3 copy number were immunoblotted for GLUT3 (encoded by SLC2A3) and it was found that the protein level was significantly correlated with the genomic copy number (p value = 0.020). Therefore, we concluded that SLC2A3 CNV is a genetic modifier of the AO in HD, associated to a variance of the disease onset and affecting the gene expression. To investigate the functional basis of this effect, we analysed lines showing over- or under-expression of the functional homolog of SLC2A3 (Glut1) in a Drosophila model of HD. After analysing several disease-relevant metrics, including neurodegeneration of the photoreceptors, eclosion rate and longevity, we found that gain and loss of Glut1 expression can delay and worsen, respectively, neurodegeneration in HD flies. In conclusion, β-defensin genomic copy number is not associated with modulation of HD AO. On the other hand, SLC2A3 CNV is a genetic modifier of the AO in HD, and likely has functional consequences, based on our findings in patient cells and in a Drosophila HD model.