Discovery of a human VNTR allelic variant in Nprl3 gene intron that enhances its transcription in peripheral blood

Parkinson's disease (PD) is a slowly progressive degenerative disorder of the central nervous system that is classically defined in terms of motor symptoms consequent to degeneration of specific subsets of mesencephalic dopaminergic (DA) cells within substantia nigra (SN) pars compacta. No pharmacological treatment is currently available to slow or arrest the neurodegenerative process. Furthermore, accurate early diagnosis suffers from the lack of reliable biomarkers. By the time motor symptoms appear, PD patients have already lost 60-70% of DA-producing cells (Dauer & Przedborski 2003) proving that sporadic PD is diagnosed many years after the onset. It is therefore reasonable to expect that potential pharmacological treatments could be more effective if patients can benefit from it in the premotor phase. Given the systemic nature of the disease, it is not surprising that many alterations of blood physiology have been described in PD patients (Kim et al., 2004; Shults and Haas, 2005; Bongioanni et al., 1996; Migliore et al., 2002; Petrozzi et al., 2002; Salman et al., 1999; Larumbe et al., 2001; Bessler et al., 1999). In this context, a blood test to predict PD would impact the ability to identify new treatments for this incurable disease. Furthermore, it could be applied to a large number of individuals since blood is commonly used in diagnostics for being easily accessible. Gene expression analysis is a powerful tool to study complex diseases such PD and it has been extensively employed to find peripheral biomarkers (Papapetropoulos et al., 2007). In the laboratory of Prof Gustincich, in collaboration with Dr Carninci at RIKEN, Yokohama, Japan, nanoCAGE technology has been previously used to find alterations in the blood transcriptome of 20 drug naïve de novo PD patients compared to 20 Healthy Controls (HC). NanoCAGE allows the identification of Transcription Start Sites (TSSs) and therefore of the associated promoters providing an unbias quantitative description of the cellular transcriptome targeting virtually any RNA molecule present in the sample. The most up-regulated nanoCAGE tag in PD patients is located in the third intron of the gene Nitrogen Permease Regulator Like Protein 3 (Nprl3). Nprl3 gene lies on the telomeric region of human chromosome 11 and contains in its intron the major regulator elements of α globin (Hughes et al., 2005). Neklesa and Davis in 2009 (in yeast) and Bar-Peled et al. in 2013 (in mammals) proved that Nprl3 is a component of a protein complex that inhibits mTORC1 activity. In eukaryotes TOR is the major sensor of nutrients, energy and stress. Alterations in its pathway have been correlated with diseases and conditions where growth and homeostasis are compromised such as cancer, metabolic diseases and aging. The aim of my PhD thesis was to identify the full-length transcript associated to the nanoCAGE tag, validate it, and to test whether it may represent a peripheral biomarker of PD. Taking advantage of rapid amplification of cDNA ends (RACE) assay, I demonstrated that the tag represents an alternative Transcription Start Site of Nprl3 (TagNprl3). It is associated to a TCT motif (YC+1TYTYY) for initiation of transcription, which has been found to be specific for ribosomal protein coding genes and those involved in protein synthesis. The tag maps to a 29nt minisatellite that is found repeated 16 times in the reference genome. High-tag expression is associated to an allelic genomic variant of 13 repeats. To our knowledge this is the first time that a minisatellite variant is both a TSS and an expression quantitative trait locus (eQTL). Unfortunately, high TagNprl3 expression resulted not to be correlated to PD but to heterozygosity. Furthermore, allelic frequencies were not correlated to PD. I then showed that TagNprl3 is expressed in red blood cells (RBCs) both at mRNA and protein levels giving rise to an isoform truncated at the N-terminal. This is able to interact with its protein partner Nprl2 and its overexpression inhibits cell proliferation. This work provides hints for Nprl3 protein function in blood and may suggest a testable hypothesis linking mTOR activity to genomic polymorphisms in modifier genes