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Theses

Functional genomics characterization of genetic predisposing factors in uveal melanoma

Abstract : Uveal melanoma (UM) is a rare tumor, yet it is the most common intraocular malignancy in adults with 500 new cases per year in France. It arises from the malignant transformation of melanocytes of the uvea, composed of the choroid (90% of cases), ciliary body (6%) and iris (4%). UM is a genetically simple tumor characterized by two major somatic driver events: mutually exclusive mutations of GNAQ, GNA11, PLCB4 or CYSLTR2, initiating tumorigenesis by constitutive activation of the Gaq signaling pathway, and a second event through mutually exclusive mutations that define metastatic risk and patient outcome: loss-of-function mutations of BAP1 associated with its bi-allelic inactivation by monosomy 3 (M3) leads to UM with high metastatic risk and poor prognosis, while disomy 3 (D3) is associated with either SF3B1 or EIF1AX mutations and respectively confer intermediate and low metastatic risk. Prognosis is dismal after occurrence of metastasis in 30-50% of cases, almost invariably to the liver, with a median survival of ∼12 months. Strikingly, individuals of European ancestry with light eyes and fair skin are particularly at risk of developing UM, with a relative risk (RR) of up to 20 in populations of European ancestry compared to those of African or Asian ancestries. However, the absence of ultra-violet (UV) mutational signature and stable UM incidence over the past decades rule out a role for pigmentation protecting against UV light to explain this peculiar epidemiology. Besides, familial cases of UM are seen in 1% of UMs, yet germline mutations of BAP1 are the only strongly predisposing genetic risk factor known so far and only explain a fraction of familial cases, suggesting that genetic risk of UM mostly remains unaccounted for.We hypothesized that some susceptibility alleles could predispose populations of European ancestry to UM. We sought to investigate the genetic predisposing factors in UM following two approaches: (i) a candidate-gene approach by targeted-sequencing of MBD4 in a large consecutive cohort of 1,093 UM patients, allowing us to demonstrate that MBD4 is a UM predisposing gene associated with high-risk M3 UM, conferring a RR = 9.2 of developing UM compared to general population; and (ii) a genome-wide association study in UM followed by a functional genomics study to identify common, low-penetrant UM risk alleles. Through this second approach, we confirmed the UM risk region at chromosome (chr) 5 on the TERT/CLPTM1L locus and further identified two pigmentation susceptibility loci, HERC2/OCA2 (ch15) and IRF4 (chr6). Importantly, these two loci were differentially associated with high-risk, M3 and low-risk, D3 UMs respectively, distinguishing two UM subgroups marked by specific genetic risk factors influencing tumor biology and metastatic potential. Lastly, we functionally characterized the TERT/CLPTM1L UM risk locus. After demonstrating an allele-specific regulation of gene expression at this locus, we narrowed down the genomic region to one functional variant, rs452384, which exhibited allele-specific nuclear factor binding properties. Using a quantitative proteomic approach, we evidenced binding of NKX2.4 transcription factor specifically to the rs452384-T allele. We further show that knockdown of NKX2.4 in UM cell lines results in a subtle yet significant increase in TERT and CLPTM1L expression, representing the first steps towards deciphering the tumorigenic mechanism of this risk locus in UM. Finally, we suggest that rs452384 may mediate its activity through differential telomere length regulation, potentially arising prior to UM tumorigenesis. Taken together, these results shed light on multiple moderate and low-risk genetic predisposition factors in UM, which bring new insights on the underlying biological mechanisms in UM predisposition, and has clinical relevance as patients with MBD4 deficiencies may respond to immunotherapy, resulting in the addition of MBD4 to clinical genetics panels.
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Submitted on : Tuesday, February 8, 2022 - 10:57:08 AM
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Anne-Céline Derrien. Functional genomics characterization of genetic predisposing factors in uveal melanoma. Biotechnology. Université Paris sciences et lettres, 2021. English. ⟨NNT : 2021UPSLS083⟩. ⟨tel-03561357⟩

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