Personalized medicine in diabetes: Unraveling the disease casualty of HNF1A and HNF4A gene coding variants of “unknown clinical significance” in two large Norwegian diabetes registries

PhD progress rapport for 2018 by Alba KaciThis report represents my progress as a PhD candidate from December 2017 to December 2018. It also gives a summary of my PhD project, describing some of the scientific results that have been achieved so far.Progress in the research project: 1. Functional characterization of ‘variants of unknown significance’ in HNF1A/4A identified in the Norwegian MODY registry: The aim of this study was to investigate possible pathogenic effects of 16 HNF1A variants and 15 HNF4A variants of unknown clinical significance identified in the Norwegian MODY Registry, using functional protein analyses, and to correlate findings with family history and clinical characteristics of HNF1A/4A variant carriers. So far, I have carried out all functional protein assays in order to investigate a possible pathogenic effect of the 16 HNF1A variants. Based on the results of the functional investigation, 3 out of 16 HNF1A variants investigated demonstrated significantly impaired protein function and were re-classified as likely pathogenic/pathogenic. Only 1 out of 14 HNF4A missense variants was shown to have an impaired a protein function and was subsequently re-classified as likely pathogenic. Our next approach in this project is to correlate the findings of this functional evaluation with clinical characteristics of HNF1A/4A variant carriers. Potentially, this will give rise to an updated Norwegian MODY registry, which is then expected to improve diagnosis. In September (2018), a clinician was involved to gather more clinical information on the HNF1A/HNF4A variant carriers. At the same time, we have already started working in the manuscript, which we aim to finish by the end of January (2019). 2.Characterize 17 novel HNF1A variants identified in the Czech population by functional protein assays: An early and correct diagnosis of MODY is highly beneficial for the patients and their family members, thus much effort has been put into identifying new variants that cause MODY. Recently, a clinical investigation in 19 Czech families suspected to have MODY, identified 17 novel HNF1A variants that have not been previously reported in any other population studies. Together with Dr. Jana Malikova at Charles University in Prague we were able to share our expertise on functional characterization of HNF1A variants and we collaborated on a study to functionally investigate the potential damaging effect of these 17 novel HNF1A variants identified in the Czech Republic. Our functional investigations indicated that several of the novel HNF1A variants from the Czezh families resemble likely pathogenic/ pathogenic MODY3 variants in functional effect. Thus, the carriers of these variants may benefit from oral agents such as sulfonylurea rather than insulin injects. We are currently doing the final corrections to this manuscript, and it will be submitted it shortly to the Diabetes Care journal. I will have a shared first authorship on this paper. 3.Investigate new molecular mechanism describing HNF-1A transcription factor function that may lead to new diabetes treatment strategies: Although mutations in the HNF1A gene are the most prevalent inherited type of diabetes, the precise mechanism for how its gene product, HNF-1A is regulated, is largely unknown. Recent reports highlight the importance of balanced post-translational modifications of proteins in insulin-producing β-cells for maintenance of glucose homeostasis, and in particular the role of SUMOylation. Thus, we investigated whether SUMOylation could be involved in the regulation of HNF-1A and the functional relevance of HNF-1A SUMOylation in various cell models. This study is completed and we were able to identify that HNF-1A is a substrate of SUMOylation, and that a E3 SUMO ligase, PIASy, is a new interacting partner. Publications list: 1. Kaci, A., Keindl, M., Solheim, M., Njølstad, PR., Bjørkhaug, L., Aukrust, I. The E3 SUMO ligase PIASγ is a novel interaction partner regulating the activity of diabetes associated hepatocyte nuclear factor-1α. Sci Rep. 2018 Aug 24;8(1):12780. doi: 10.1038/s41598-018-29448-w. 2. Malikova*, J., Kaci*, A., Dusatkova, P., Aukrust, I., Torsvik, J., Dvorakova, P., Vesela K., Njølstad, PR., Pruhova, S and Bjørkhaug, L. Functional analysis of Czech HNF1A-MODY variants refines classification of predicted variant pathogenicity. *Co-first authors (manuscript in preparation). 3. Kaci, A., Svalastoga P., Molnes, J., Tjora, E., Johansson, B., Bjørkhaug, L., Aukrust, I., and Njølstad, P. Functional characterization of HNF1A/HNF4A variants identified in the Norwegian MODY registry can be important for precision medicine in diabetes clinics. (manuscript in preparation).

PhD progress rapport for 2017 by Alba KaciThis report represents my progress as a PhD candidate from December 2016 to October 2017. It also gives a summary of my PhD project, describing some of the scientific results that have been achieved so far.My PhD project aims to functionally characterize HNF1A and HNF4A variants of “uncertain clinical significance”, in the two Norwegian Registries, and to correlate the findings with clinical characteristics of HNF1A/HNF4A variant carriers. In addition, my project aims to investigate new mechanisms for the regulation of the HNF-1A protein, such as post translational modification (SUMOylation). Progress in the research project: 1. Functional characterization of HNF1A/4A variants: To date there are 44 missense HNF1A variants and 11 missense HNF4A variants in the MODY Registry. Of these are 26 of HNF1A and 7 of HNF4A of unknown significance. In the Norwegian Childhood Registry there are 13 missense HNF1A variants and 4 missense HNF4A variants. Of these 9 HNF1A and all of HNF4A variants are of unknown significance. So far, I have carried out functional protein assays in order to investigate a possible pathogenic effect of the 15 HNF4A missense variants identified in the Norwegian MODY Registry and the Norwegian Childhood Diabetes Registry. Interestingly, I was able to identify one HNF4A variant found in both registries and classified as of unknown significance to be likely pathogenic. In addition, I have started designing all the HNF-1A unknown significance variants by site directed mutagenesis and will validate them by Sanger sequencing. After all the constructs are ready, functional assays will be carried to examine their pathogenesis. 2. SUMOylation: So far, I have data showing for the first time that HNF-1A is a substrate for SUMOylation both in target cells and in vitro. I have also found three SUMOylation sites in HNF-1A and shown that SUMOylation affects both the activity and stability of HNF-1A. Moreover, I have recently identified protein inhibitor of the activated STAT (PIAS) as a novel HNF-1A interaction partner which leads to destabilization of HNF-1A by nuclear translocation and subsequent transcriptional repression. This summer, I was able to present those findings in two international conferences: the American Diabetes Association’s 77th Scientific Session (San Diego) and the Ubiquitin and SUMO: from molecular mechanisms to system-wide response (Dubrovnik). Progress in the training component: So far, I have obtained 24 credits. Thereby I need only 6 credits until my training component is completed. To achieve this I am planning to obtain 1 credit during my midway evaluation next spring (2018) and the rest of the credits on the animal course organized by the university. Publications published: Since my last progress report, I had a manuscript in preparation based on the findings in the SUMOylation project which was planned to be submitted during spring 2017. However due to our new interesting data which strength our findings we chose to work more on the manuscript. The manuscript is now almost done and we are working on the last revision. Our plan is to first submit at Cell Metabolism at the end of January 2018. Regarding the other part of my project, we aim to publish our findings within the end of 2018 and beginning of 2019.

Unraveling the disease casualty of HNF1A and HNF4A gene coding variants in two large Norwegian diabetes registriesThis report represents my progress as a PhD candidate from April 2016 to December 2016. It also gives a summary of my PhD project, describing some of the scientific results that have been achieved so far.Maturity-onset Diabetes of the Young (MODY) is a monogenic form of diabetes which represents 2-3 % of all diabetes cases in Norway. Mutations in the genes encoding the transcription factors hepatocyte nuclear factor 1-alpha (HNF-1A, MODY3) and hepatocyte nuclear factor 4-alpha (HNF-4A, MODY1) are two of the most common MODY forms. Genetic testing is usually performed to identify previously reported genetic variants associated with the disease, and thereby the diagnosis of MODY is set. However, genetic variants that so far have not been associated with MODY and are not well described in the literature are also identified during genetic testing. These are then classified as variants of unknown significance. My PhD project aims therefore to functionally characterize HNF1A and HNF4A variants of “uncertain clinical significance”, in the two Norwegian Registries, and to correlate the findings with clinical characteristics of HNF1A/HNF4A variant carriers. This will be then used to verify a genetic diagnosis and allow alternative treatment strategies for these affected patients. To date there are 44 missense HNF1A variants and 11 missense HNF4A variants in the MODY Registry. Of these are 26 of HNF1A and 7 of HNF4A of unknown significance. In the Norwegian Childhood Registry there are 13 missense HNF1A variants and 4 missense HNF4A variants. Of these 9 HNF1A and all of HNF4A variants are of unknown significance. So far, I have carried out functional protein assays in order to investigate a possible pathogenic effect of the 15 HNF4A missense variants identified in the Norwegian MODY Registry and the Norwegian Childhood Diabetes Registry. Interestingly, I was able to identify one HNF4A variant found in both registries and classified as of unknown significance to be likely pathogenic. This spring, I am planning therefore to present my findings at the EASD study Group on Genetics of Diabetes meeting which will be organized in Leiden, May 11th - 13th, 2017. Ongoing studies are also carried out at the main to investigate other protein aspects affected by those variants. My future plan is to prepare a manuscript based on the findings and submitted preferably after this summer (fall 2017). In addition, my project aims to investigate new mechanisms for the regulation of HNF-1A, such as post translational modification (SUMOylation). So far, I have data showing for the first time that HNF-1A is a substrate for SUMOylation both in cellullo and in vitro. I have also found three SUMOylation sites in HNF-1A and shown that SUMOylation affects both the activity and stability of HNF-1A. All the gathered data indicate thus that SUMOylation of HNF-1A represents a novel post translational regulatory mechanism of HNF-1A in pancreatic ß–cells, most likely with implication for glucose-stimulated insulin secretion. Last fall, I was able to present those findings in two international conferences: the 52nd EASD Annual Meeting (Munich) and the Copenhagen Bioscience Conference, Protein Signaling- From pathways to Networks. The feedback I received at the conferences helped me to complete some of my experiments and strengthen my findings. I have a manuscript in preparation based on these findings ( Kaci et al., The transcriptional activity of the hepatocyte nuclear factor 1 alpha is regulated by the protein inhibitor of activated STAT (PIASy) ) which I plan to submit for publication at the beginning of this year (spring 2017).