Modeling Monogenic Diabetes Mody3 Using Human Pluripotent Stem Cells

Understanding monogenic diabetes has been challenging due to the lack of human model but also because mouse models do not recapitulate the disease. Here, we use human pluripotent stem cells (ESCs) and CRISPR-CAS9 (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9) nucleases to understand the most common form of monogenic diabetes, Maturity Onset Diabetes of the Young 3 (MODY3) caused by heterozygous mutations in the transcription factor HNF1A. In this thesis research, we found that the lack of HNF1A does not impair the generation of beta-like cells; Wild-type (WT) and mutant cell lines presented similar differentiation efficiency between genotypes. HNF1A is necessary to repressed alpha cell fate; loss of HNF1A leads skew beta-like cell differentiation towards alpha cell type. We also explore beta cell function related to insulin secretion and cellular bioenergetics; we found that HNF1A needs it for proper insulin secretion and cellular respiration leading to lower glycolysis metabolism and mitochondrial respiration. We explore the HNF1A relationship between phenotype and genotype by generating a genome profiling analysis of HNF1A. We MODY3 models confirmed that many of the genes that are dis-regulated in the Hnf1α mouse model such us HNF4A, PCSK1, GC, DDP4, KIF12 6GPC2 and others. Besides, we also found a large number of genes to be uniquely dis-regulated in the human model, which could explain the species-specific phenotype. These genes include the transcription factors RFX6, GLIS3 and PAX4, the metallothionein gene family a novel long non-coding (lnc)RNA LINC01139. This lncRNA is only present in primates, and also it is expressed in human endocrine cells. We interrogate the role of this lncRNA, and we found that it recapitulates a subset of phenotypes seen in HNF1A, related with defects in glycolysis, mitochondrial respiration, and downregulation of group of genes downstream of HNF1A. Our work offers a model to define the unique biology of HNF1A in humans that may be distinct from rodent models leading to a better understanding of human beta cell physiology with clinical implications for MODY3 patients but also with broader applications to diabetes

Forkhead box R1-mediated stress response linked to a case of human microcephaly and progressive brain atrophy

Forkhead box (Fox) family transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. This family was named after the ectopic head structures observed in mutants of the Drosophila gene forkhead (fkh). Since the discovery of fkh, hundreds of Fox genes have been identified in organisms ranging from yeasts to humans, making it one of the largest but least explored families of higher eukaryotic transcription factors. The NIH Undiagnosed Diseases Program (NIH UDP), a clinical site of the NIH Undiagnosed Diseases Network (UDN), identified a variant (p.M280L) in a single allele of the FOXR1 gene in an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy, and global developmental delay. The de novo missense variant in FOXR1 converts a highly conserved methionine residue at amino acid 280 to leucine and was predicted to contribute to the individual’s disease. The goal of this research is to investigate the biological role of FOXR1 and to determine how the M280L mutant leads to disease pathogenesis. At the protein level, the M280L mutant impaired FOXR1 expression and induced a nuclear aggregate phenotype when overexpressed in HEK 293T and COS7 cells due to protein misfolding and proteolysis. A FOXR1 C-terminal truncation mutant mimicked the M280L phenotype, suggesting that the C-terminal sequences of FOXR1 are important for FOXR1 protein stability. RNAseq and pathway analysis in HEK 293T cells indicated that FOXR1 acts as both transcriptional activator and repressor, playing central roles in heat shock response, chaperone cofactor-dependent protein refolding, and cellular response to stress. Indeed, FOXR1 expression is increased in HEK 293T in response to cellular stress, a process in which FOXRI directly controls HSPA6, HSPA1A and DHRS2 transcription. In contrast, the ability of the M280L mutant to respond to stress is compromised, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Combined, these results suggest that FOXR1 plays a role in cellular stress and that impairment of these functions may contribute to the disease phenotypes seen in the individual with the FOXRI M280L variant

The role of non-coding genetic variants on transthyretin gene transcription in transthyretin amyloidosis

The transthyretin-associated amyloidoses are a group of protein-folding disorders caused by deposition of the liver-secreted plasma protein transthyretin (TTR) in various tissues of the body. The sporadic form of the disease is caused by deposition of wild-type TTR whereas the inherited form is caused by deposition of mutated TTR; there are over 100 known amyloidogenic mutations of the TTR gene. The transcriptional regulation of the mouse transthyretin gene has been well studied. Organ-specific modulation of TTR mRNA is achieved by coordinated binding of hepatocyte-specific and ubiquitously expressed transcription factors to regulatory regions in the proximal promoter and upstream enhancer region. The hypothesis of this dissertation is that non-coding genetic sequence variations in the promoter of the transthyretin gene situated upstream of the regulatory regions, alter its transcriptional regulation, contributing to the onset and expression of transthyretin-associated amyloidosis. Previously, we identified a significant association of a non-coding polymorphism of the TTR promoter, rs3764479, with age of onset and survival in patients with ATTRwt amyloid disease. In this dissertation, electrophoretic mobility shift assays (EMSA) were used to investigate transcription factor binding of HepG2 nuclear proteins to short DNA probes with and without rs3764479. These mobility shift studies revealed that HepG2 nuclear extract proteins showed higher affinity to the wild-type TTR sequence than to one containing the rs3764479 SNP. Competition EMSAs suggested SNP-related changes in the binding of transcription factors hepatocyte nuclear factor-1 (HNF1) and hepatocyte nuclear factor-3b may alter TTR gene transcription. To investigate transthyretin gene regulation in V122I ATTR amyloid, the most prevalent TTR gene variant in the United States, the proximal promoter region from patients with V122I ATTR amyloidosis was sequenced and analyzed. In total, 8 SNPs were identified; one (rs955705399) was significantly associated with disease between the two V122I genotype-positive cohorts studied with and without cardiomyopathy. It is postulated that the presence of SNPs could influence gene expression and ultimately disease pathogenesis. In summary, these studies suggest that presence of disease-associated non-coding genetic variations modify transthyretin gene expression by disrupting transcription factor binding and may, in part, explain the clinical heterogeneity seen in patients with transthyretin-associated amyloidoses

Exploring the Grey Zone between Type 1 and Type 2 Diabetes

T1D is most common in children and young adults and is characterized by autoimmune destruction of insulin producing pancreatic beta cells, presence of certain risk genotypes such as HLA-DQB1, INS VNTR, PTPN22 and need of insulin for survival. In adults the same situation is often referred to as Latent Autoimmune Diabetes in Adults (LADA), with age at onset after 35 years and non-insulin requiring at least for 6 month after diagnosis. On the other hand, T2D is characterized by impaired insulin secretion and/or insulin resistance, which coexists with excessive hepatic glucose production and abnormal fat metabolism. Environmental factors causing insulin resistance are puberty, pregnancy, weight gain (central obesity “apple type”) and sedentary lifestyle. Usually T2D is diagnosed after 40 years of age and in some cases is diagnosed when patients develop vascular and neuropathic complications. TCF7L2 is by far the strongest T2D-associated gene. Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes inherited in an autosomal dominant fashion (individual has one copy of a mutant gene and one normal gene on a pair of autosomal chromosomes) characterized by nonketotic diabetes, age at onset before 25 years and primarily defect in beta-cell function. Until now, mutations in six genes have been identified as the cause of different forms of MODY, i.e. HNF-4 (MODY 1), glucokinase (GCK) (MODY 2), HNF-1 (MODY 3), IPF1 (MODY 4), HNF-1ß, formerly TCF2 (MODY 5) and NeuroD1 (MODY6). The goal of this thesis was to genetically dissect autoimmune (T1D and LADA) and non-autoimmune (T2D and MODY) diabetes in young (15-34 years old)and middle-aged (40-59 years old) Swedish diabetic patients for proper diagnosis and treatment of the disease in the future. To fulfill our goals we have selected 1642 young (15-34 years old) adult diabetic patients from Diabetes Incidence Study in Sweden (DISS) and 1619 middle-aged (40-59 years old) diabetic patients from Diabetes Registry in Southern Sweden. We determined genetic markers: HLA-DQB1 (study I and II), PTPN22, Ins VNTR, TCF7L2 (study II), PPARG, KCNJ11, IGF2BP2, WFS1, CDKAL1, JAZF1, CDKN2A/2B, HHEX, SLC30A8 and FTO (study III) and MODY genes- HNF-4 , GCK, HNF-1 and HNF-1ß, formerly TCF2 (study IV), measured islet antibodies (ICA, IA-2A and GADA) and C-peptide (marker of beta-cell function instead of insulin). In Study I we evaluated whether HLA-DQB1 genotypes facilitates the classification of diabetes as compared with islet antibodies among young (15-34 years) adult diabetic patients. Islet antibodies were found among 83% clinically considered to have T1D, 23% with T2D and 45% with unclassifiable diabetes.fpC-peptide concentrations after diagnosis were markedly lower in patients with than in those without islet antibodies. Irrespective of clinical classification, patients with islet antibodies showed increased frequencies of at least one risk HLA-DQB1 genotypes compared with patients without. Antibody negative patients with risk HLA-DQB1 genotypes had significantly lower fasting fpC-peptide concentrations than those without risk genotypes. We concluded that Assessment of islet antibodies is necessary for an etiological classification of diabetic patients. HLA-DQB1 genotyping does not improve the classification in patients with islet antibodies. However, in patients without islet antibodies, HLA-DQB1 genotyping together with fpC-peptide measurement may be of value in the differentiation between idiopathic T1D versus T2D. In Study II we evaluated whether genetic markers associated with T1D (HLADQB1,INS VNTR and PTPN22) and T2D (TCF7L2) could help to discriminate between autoimmune and non-autoimmune diabetes in young (15-34 years) and middle-aged (40-59 years) diabetic patients. Frequency of risk genotypes HLADQB1, PTPN22 CT/TT, INS VNTR class I/I and INS VNTR class IIIA/IIIA was increased in young and middle-aged GADA+ compared with GADA- patients. T2D-associated genotypes of TCF7L2 CT/TT of rs7903146 were significantly more common in young GADA- than in GADA+ patients. No such difference was seen in middle-aged patients, in whom the frequency of the CT/TT genotypes of TCF7L2 was similarly increased in GADA- and GADA+ groups. We concluded that common variants in the TCF7L2 gene help to differentiate young but not middle aged GADA+ and GADA- diabetic patients, suggesting that young GADA- patients have T2D and that middle-aged GADA+ patients (LADA) are different from their young GADA-positive (T1D) counterparts and share genetic features with T2D. In Study III we genotyped a panel of 10 novel T2D-associated risk genotypes in young (15-34 years) and middle-aged (40-59 years) GADA+ and GADA- diabetic patients and evaluated how they would modify the clinical phenotype. Young GADA- patients had increased frequency of risk variants in the PPARG, IGF2BP2, WFS1, JAZF1 and CDKN2A/2B genes compared with an elderly nondiabetic control group. Also risk variants in JAZF1 (AA) and CDKN2A/2B (TT) were more common in GADA- than in GADA + young diabetic patients. As expected middle-aged GADA- patients had increased prevalence of risk variants in the PPARG, IGF2BP2, WFS1, CDKAL1, JAZF1, SLC30A8, CDKN2A/2B, KCNJ11 and FTO genes compared with non-diabetic controls with no significant difference compared with GADA+ patients. Middle-aged GADA diabetic patients with more risk alleles (≥12) had decreased C-peptide concentrations than patients with less risk alleles (≤9). Also, GADA+ patients with more risk alleles had an earlier age at onset than GADA+ patients with less risk alleles. Distribution of T2D-associated risk alleles was quite similar inmiddle-aged patients regardless of presence of GADA. T2D- associated risk genotypes modify the disease phenotype (age at onset and C-peptide) in middleaged but not in young diabetic patients. In Study IV we evaluated whether common variants in MODY genes can discriminate between autoimmune and non-autoimmune diabetes in young adult diabetic patients and screened antibody negative diabetic patients with 3 members with diabetes in the family for HNF-4 , GCK and HNF-1 mutations. No significant difference in frequency of common variants in MODY genes was seen between Ab+ and Ab- individuals. In Ab+ diabetic patients carriers of the T2D-associated T allele of the HNF-1 gene had higher age at onset of diabetes, but severe symptoms of diabetes (weight reduction and/or polyuria) than G allele carriers. Finally, in Ab- diabetic patients carriers of the T2D-associated G allele of HNF-1ß gene had less frequent weight reduction and/or polyuria and ketonuria at diagnosis than A allele careers. One patient had frameshift mutation in exon 4 designated “Pro291fsinsC” in the HNF-1 gene. Common variants in MODY genes do not discriminate between young patients with autoimmune and non-autoimmune diabetes but they do influence onset and presentation of the disease. Our studies show that genetic markers clearly improve the classification of diabetes and together with islet antibodies they might be of help for diagnosis and treatment of different diabetic subgroups

The effect of dietary compounds on human cathelicidin antimicrobial peptide gene expression mediated through farnesoid X receptor and its potential role in gastrointestinal health

The human cathelicidin antimicrobial peptide (CAMP) is a broad spectrum microbicidal agent and modulator of both the innate and adaptive immune system. It is induced by 1,25-dihydroxyvitamin D (1,25(OH)₂D₃) through activation of the vitamin D receptor (VDR) and primary bile salts through activation of the xenobiotic nuclear receptor farnesoid X receptor (FXR). Both receptors are expressed by enterohepatic and gastrointestinal (GI) tissues and play important roles in GI immunity and homeostasis. It has been demonstrated by us and others that plant polyphenol xanthohumol (XN) acts as an FXR ligand, but its regulation of CAMP gene expression has not been determined. We hypothesize that plant polyphenols obtained in the diet act as ligands for FXR and regulate expression of the CAMP gene in the GI tract thereby promoting gastrointestinal health through improved barrier defense against infection and inflammation. In this study, we demonstrate that XN induces BSEP (bile acid export pump) and human CAMP promoter activity via FXR. This activation appears to require some combination of the vitamin D response element (VDRE) site in the CAMP promoter and a potential FXR response element (FXRE) located in the third exon of the gene. In addition, XN and its metabolite 8-prenylnaringenin (8-PN) induced the mRNA expression of several FXR target genes including: BSEP, SHP (small heterodimer partner), IBABP (ileal bile acid binding protein), CAMP and FXR in biliary carcinoma cell lines. Combinations of 1,25(OH)₂D₃ and either XN or 8-PN cooperatively induced endogenous CAMP gene expression in cells. We conclude that the plant polyphenol XN and its metabolite 8-PN act as FXR ligands and regulate expression of the human CAMP gene alone or in combination with 1,25(OH)₂D₃. A second distinct project used immunohistochesmitry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) to characterize a transgenic mouse that carries the human CAMP gene. In the transgenic mice fed a normal diet, the human CAMP gene is expressed in immune and epithelial barrier cells and treatment of tissues with 1,25(OH)₂D₃, including those from the GI tract, induced expression of the human, but not the mouse gene. This study advances our understanding of human CAMP gene regulation by FXR and also helps establish the mouse as a reliable model system that can be used in future studies to determine the importance of CAMP gene expression in human health

Deciphering the role of BMP4 signalling and gene regulation in the specification of human liver progenitor cells

Early hepatic specification and organogenesis can be modelled in vitro using human induced pluripotent stem cells (hiPSCs). These models apply differentiation protocols to direct hiPSCs through all the key developmental stages to accurately reflect in vivo development. Bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signalling are crucial for the specification of hepatic progenitors during early liver development. While the signalling cascades of these two morphogens are well characterized, the mechanisms by which they promote hepatic cell fate choice and hepatic gene expression in anterior foregut endoderm (FE) cells is not very well understood. In this project, we characterize hiPSCs-based model of early liver development and apply it to understand the role of BMP signalling in hepatic specification. We confirm that BMP4 signalling is also necessary for liver progenitor cells (LPCs) specification from FE during hiPSCs differentiation. Using RNA sequencing (RNA seq.) we examine transcriptome changes induced by BMP4 during the transition from FE to LPC stage. Overrepresentation analysis (ORA) and gene set enrichment analysis (GSEA) analysis revealed early activation of hepatocyte-specific functions such as lipid and protein homeostasis, haem metabolism or coagulation, while at the same time, cell adhesion and locomotion related genes are downregulated indicating preparation for cell migration out of the forming liver bud. We also notice upregulation of all four FGF receptors upon BMP signalling indicating at possible cross talk between the two pathways. The RNA seq. also detected a number of BMP4 upregulated transcription factors (TFs), several of these TFs are known for their roles in multiple developmental processes. Among them TBX3, previously reported to have a role in hepatic specification in mice, and two other TBX family members: TBX2 and TBX20. As a preliminary screen, we used a published, optimized protocol for creating inducible knockdown hiPSC lines to assess the importance of TBX and other TFs for the process of LPC specification. Double knockdown of TBX3 and TBX20 TFs significantly disrupted the hepatic induction process as shown by decreased expression of early hepatic genes such as TTR, AFP, AAT and ALB. Further studies are necessary to confirm and further characterize the role of TBX TFs for hepatic specification. Our study demonstrates that application of hiPSCs derived models for the study of development can aid the understanding of molecular mechanisms driving early liver specification and improve our understanding of human embryology and organogenesis. This knowledge can also be used to created more efficient differentiation platforms that can yield more mature, functional and clinically relevant populations of hiPSC-derived hepatocytes