Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disorder characterized by motor, psychiatric and cognitive dysfunction. HD symptoms appear in mid-life, with irreversible progression over 10-25 years. The disease is characterized by focused neuronal vulnerability with predominant loss of striatal and cortical neurons. The disease is caused by an expansion of the CAG repeat in the exon 1 of the huntingtin gene (HTT), which leads the production of mutant HTT protein (muHTT) with an elongated polyglutamine (polyQ) stretch (Cattaneo et al., Nat Rev Neurosci. 2005; Zuccato et al., Physiol Rev. 2010). There is an inverse correlation between the CAGs lengths and the age at the disease onset, i.e. the longer the CAG, the earliest the symptoms. Individuals with 35 or fewer repeats do not develop the disease, whereas those with 40 or more repeats (fully penetrant alleles) are invariably affected (Hauck B., 2003).
In humans, the CAG tract in HTT is highly unstable and prone to expansion, especially during paternal transmission (Merritt, 1969). Pathological repeats can expand further during development to generate a mosaic of cells with differing repeat lengths. In fact, increases in the number of CAG repeats have been observed in mitotically dividing cells throughout the lifetime of HD individuals (McMurray, 2010). Analysis of post- mortem brain tissues from HD patients has revealed high mosaicism in CAG size and very large expansion also in non-proliferating tissues such as the striatum and cortex (Telenius, 1994; De Rooij, 1995; Wheeler, 1999). This phenomenon has been described also in HD models.
The expansion of the CAG tract has been well documented in brain tissue from HD mice (Mollersen, 2010; Gonitel, 2008; Larson, 2015). In particular, current evidence shows that the striatal neurons are characterized by a high rate of CAG repeat allele instability compared to other neuronal types, suggesting that functional polymorphisms can be produced in adult neurons. This aspect can also be an intriguing explanation for age-of-onset variability observed between individuals carrying similar germinal CAG length (Larson, 2015). Altogether, this evidence indicates that CAG expansions occur in post-mitotic neurons and may continue during lifetime of the individual and contribute to exacerbate neuronal toxicity and selective neuronal degeneration. The fact that significant CAG repeats length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating somatic mutations in brain tissue (Shelbourne, 2007). Somatic repeat variation may also occur during the many billions of mitoses characterizing normal brain development (Nithianantharajah, 2007).
CAG instability occurs also in long-term cultured fibroblasts from HD mice already after 11 passages (Manley, 1999), in cultured human astrocytes (Farrell and Lahue, 2006), and in neurons derived from pluripotent stem cells (Niclis, 2009).
This project aims to identify the genetic factors that are implicated in HTT CAG instability both during mitotic cell replication and in post-mitotic neurons. We will employ an unbiased discovery process that relies on a new human embryonic stem (hES) cell-based platform, barcoded DNA libraries, CRISPR technologies and long-read third generation DNA sequencing. Factors modulating CAG elongation have been divided into cis-acting elements (i.e. DNA sequences in the proximity of the repeat or the repeat itself) and trans-acting elements (i. e. other genes non-proximal to HTT) whose interaction with the repeat contributes to its instability (Richards, 1994). The identification of genetic elements that may contribute to the CAG instability is of pivotal importance, considering the large effect they represent in the disease manifestation, and would expand the pool of available therapeutic targets with the aim of mitigating CAG expansion and therefore, disease symptoms.
