Objective
Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-secreting beta cells by autoreactive T cells. Both genetic and yet unknown factors contribute to initiation of islet autoimmunity and progression to T1D. Our research has led to the discovery of over 100 T1D risk loci in the human genome, with the vast majority of risk variants located in regions predicted to regulate gene expression in immune system related cells (particularly, CD4+ T cells). It is apparent that once an individual has two or more islet autoantibodies (markers of autoimmune targeting beta cells), it is likely the individual will progress to clinical (stage 3) T1D. At the same time, relatively little research has focused on the pre-clinical stages of T1D, particularly what factors predict the transition from the first islet autoantibody to multiple islet autoantibodies (stage 1 T1D).
Cells usually regulate gene expression through a mechanism called transcription, in which the DNA sequence of a gene is copied into a molecule called pre-mRNA that contains both coding regions (exons) and non-coding regions (introns) of the gene. Before a protein can be made, the introns are removed, and the exons are joined together in a process called ‘splicing’. Cells can rearrange the exons in different combinations, a process called ‘alternative splicing’. This allows a single gene to generate differing versions of mRNAs, each which could produce a different isoform of a protein. Our previous work showed that genetic variants in several T1D risk genes promote alternative splicing, leading to different isoform usage between individuals with T1D compared to those without T1D.
Splicing in T1D could reflect multiple biological mechanisms involving gene regulation and generate protein isoforms associated with T1D. The variation in protein isoforms is tightly regulated by RNA binding proteins (RBPs). Based on our data showing the contribution of splicing events to the risk of T1D, this proposed research focuses on mechanisms that drive progression from a single to multiple islet autoantibodies through detection of splicing events, alternative isoforms, and regulation by RBPs. These results will identify key RBP drivers of islet autoimmunity and identify those RBPs suitable for early intervention. There are ~1500 RBPs that tightly regulate alternative splicing and transcript isoform diversity, and these proteins are a rich and understudied set of candidates for T1D.
The proposed research is an Ancillary Study from The Environmental Determinants of Diabetes in the Young (TEDDY) cohort. We will use longitudinally collected peripheral blood mononuclear cells (PBMCs) and TEDDY’s immunologic, risk factor, and biomarker data from 50 participants with a single persistent islet autoantibody and 50 participants who develop multiple islet autoantibodies. We will examine alternative splicing at two time points for each individual and use long-read RNA sequencing to characterize full length isoforms in CD4+ T cells collected at each time point. Using these data, we will apply novel analytic approaches to identify candidate genes and RBPs that impact the transition from single to multiple islet autoantibodies. We will test if alternative splicing and its regulation by RBPs represents a mechanism that alters immune cell function and can be a novel target for potential intervention.
RBP dysregulation has been shown to be important in cancer and other disorders. Targeting RBPs and splicing events for intervention is now feasible through use of antisense oligonucleotides (ASOs), siRNAs, and RNA-guided protein degradation (PROTACs) to modify RBP expression or function (including FDA approved treatment of spinal muscular atrophy (SMA; Nusinersen/Spinraza) which modulates RBP-mediated splicing), making RBPs potential therapeutic targets to prevent development of a second islet autoantibody (and ultimately T1D) in those with a first-appearing islet autoantibody.
Background Rationale
Twin and family studies have shown that genetic factors account for ~50% of the risk of type 1 diabetes (T1D). Our work has led to the discovery of over 100 regions in the human genome where DNA changes are associated with T1D risk. We have shown that these changes in the DNA appear to have their effect on the regulation of the expression of their target genes and were more likely to be expressed in immune-relevant cell types (such as CD4+ T cells). While these studies have been instrumental in improving genetic risk screening for T1D, they have yet to lead to approaches that can intervene during the early stages of autoimmune attack on beta cells to prevent T1D progression.
In T1D, an unknown “trigger” causes an individual’s immune system to attack and destroy beta cells over a period of time, with a more virulent form in childhood but one that can occur at nearly all stages of life. The evidence for this attack is the presence of islet autoantibodies (IAA, GADA, IA-2A, ZnT8) found in the circulating blood. The risk of developing T1D in genetically high-risk children from a single islet autoantibody is low (10%-15%); in contrast, the risk of T1D in children with multiple islet autoantibodies is much higher (55%-70%). The modifiable risk factors that account for the transition from a single to multiple islet autoantibodies are not known, yet the time after the appearance of a first islet autoantibody is critical for intervention to prevent the occurrence of a second islet autoantibody and, ultimately, T1D.
Research suggests that a gene’s instructions are “packaged” through a mechanism called ‘alternative splicing’, and incorrect packaging can influence T1D risk. Selected segments of a gene’s sequence are packaged and used to code for a protein. Which segments are chosen is a key part of gene regulation and determines the final protein structure and function. A single gene, through alternative splicing, can give rise to many distinct protein isoforms. This diversity is necessary for cells to adapt to external signals, yet certain changes in splicing can increase an individual’s risk of disease. We have shown that some genetic variants in known T1D-associated genes can alter the process of alternative splicing. Many of these splicing differences involve regulatory sequences that affect other proteins which tightly control gene regulation (RNA-binding proteins, or RBPs). It has been shown that disrupting RBP function can lead to altered immune function and autoimmunity. We have identified ten RBPs within seven regions associated with T1D risk, suggesting that mechanisms involving alternative splicing and disrupted regulation by RBPs could be important in the development of T1D, particularly in the transition from a single to multiple islet autoantibodies.
In this study, we will use samples and data from The Environmental Determinants of Diabetes in the Young (TEDDY) study to compare participants with a single, persistent islet autoantibody (at high genetic but low total risk of T1D) with participants who progress to multiple islet autoantibodies (high for both genetic and overall T1D risk). Existing longitudinal samples collected from blood will be used to extract CD4+ T cells (a critical immune cell type) for RNA extraction (gene regulation) and process using both long-read and short read RNA sequencing to fully characterize alternatively spliced isoforms. Our strategy will uncover genes that impact T1D risk through mechanisms involving alternative splicing and disrupted regulation of RBPs. As current molecular technology has targeted RBPs for therapeutics, these studies could provide new targets for intervention in the autoimmune process, specifically at the transition from a single to multiple islet autoantibodies.
Description of Project
Type 1 diabetes (T1D) develops from the destruction of insulin-secreting beta cells in the pancreas by autoreactive T cells, with risk of T1D determined by both genetic and other (unknown) factors. We have pioneered the discovery of over 100 genetic factors contributing to risk, with most predicted to regulate expression of their target genes in immune cells (such as CD4+ T cells). The primary marker of beta cell destruction is the presence of any of four islet autoantibodies (IAA, IA-2A, GADA, and ZnT8), with the occurrence of the first islet autoantibody dependent, in part, on age and genetics. Although presence of a first islet autoantibody is not a strong predictor of T1D, the progression to a second islet autoantibody greatly increases the risk of T1D, perhaps as high as 80% risk over the subsequent 8 years. Thus, a major point for intervention to halt beta cell destruction is the period following the detection of the first islet autoantibody (prior to development of a second). The limited research conducted on this early stage of T1D has not identified modifiable factors that predict the transition from the first islet autoantibody to multiple islet autoantibodies (stage 1 T1D).
Our work has shown that regulation of gene activity in immune cells, particularly CD4+ T cells, contributes critically to T1D. In cells, protein sequences coded in DNA are first transcribed into a precursor mRNA (pre-mRNA). A mature mRNA molecule is generated by removal and joining of specific sequence segments in the pre-mRNA through a process called ‘splicing’. Cells can vary the way these segments are joined together, a process called ‘alternative splicing’ which lets a single gene to produce multiple mRNA transcripts and, as a result, different protein isoforms. Alterations in alternative splicing can shift protein production in ways that influence immune function and increase T1D risk. In our earlier work, we have shown evidence of alternative splicing in known T1D genes that separates those individuals with T1D from those without the disease. Our studies (and those of others) have not examined the full range of alternatively spliced mRNAs in the relevant immune cell type, CD4+ T cells. Understanding the complete landscape of mRNA molecules is essential, as subtle differences in protein isoforms coded by alternatively spliced mRNA can profoundly affect immune cell behavior, response to environmental triggers, and potentially drive T1D risk.
The proposed research aims to address this gap in early stages of T1D (single to multiple islet autoantibodies) by analysis of splicing in CD4+ T cells from participants of The Environmental Determinants of Diabetes in the Young (TEDDY) study. We will contrast mRNA in 50 TEDDY participants who developed a first islet autoantibody and did not progress to a second to 50 participants who developed a first autoantibody and rapidly developed a second (stage 1 T1D). TEDDY has collected blood samples over time that can be used to isolate CD4+ T cells near the time of autoantibody appearance, followed by long-read RNA-sequencing to obtain the full range of alternatively spliced mRNA isoforms. The process that generates alternatively spliced isoforms is regulated by RNA binding proteins (RBPs). Our research will identify key RBPs that could be suitable targets for early intervention. Recently, RBPs have been shown to play key roles in cancer and other diseases, prompting development of tools to alter their activity, including FDA approved drugs that modify RBP-mediated splicing. Identifying RBPs involved in the early stages of T1D would provide a novel target for halting the autoimmune process while salvaging existing insulin-producing beta cells.
Anticipated Outcome
Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-secreting beta cells by autoreactive T cells. Both genetic and (unknown) environmental factors contribute to initiation of autoimmunity and progression to T1D, and we have contributed significantly to the discovery of over 100 T1D-associated regions. The DNA sequence differences between people living with T1D from individuals without T1D are often found in regions of the genome that regulate how much of a of T1D-associated gene gene is made and which form of the gene is expressed in immune cells (e.g., CD4+ T cells)
A critical step in the development of T1D is the transition from the presence of a single islet autoantibody (an early marker of autoimmune attack on pancreatic beta cells) to presence of multiple islet autoantibodies. This transition greatly increases the risk of developing T1D. The risk of T1D in the general population without islet autoantibodies is ~0.5%, which is ~16 times less than the risk in a person having a first-degree relative with T1D (8%). In individuals with a high genetic risk, having a first islet autoantibody increases the T1D risk to 10%-15%; however, the presence of a second islet autoantibody raises the risk to 55%-70%. The transition from a single to multiple islet autoantibodies marks a critical stage in T1D development. There is little information on the molecular factors that predict this transition, which is a major research gap that we will address in this proposed study. We anticipate that this study will reveal how genetic risk for T1D changes the way immune cells process genes during the transition from one to multiple antibodies.
Our prior work has shown that T1D risk variants can affect a process called ‘alternative splicing’, a key biological mechanism for generating protein diversity from the gene code. During alternative splicing, DNA-derived RNA sequences are joined (spliced together) in a variety of combinations that generate multiple isoforms of a gene. The splicing process is tightly regulated by RNA binding proteins (RBPs) which adjusts gene expression in response to activation signals, such as those triggered during an autoimmune attack. We will determine how alternative splicing events and impaired RBP function in CD4+ T cells impact the transition from single to multiple islet autoantibodies and, ultimately, T1D.
This research is an ancillary study from The Environmental Determinants of Diabetes in the Young (TEDDY) longitudinal cohort. We will use longitudinally collected samples and rich immunologic, risk factor, and biomarker data from 50 participants with a single persistent islet autoantibody and 50 participants who develop multiple islet autoantibodies to obtain RNA from CD4+ T cells. We will perform long-read RNA sequencing to characterize full length isoforms in CD4+ T cells from each individual at two time points to identify candidate gene isoforms and RBPs that impact the transition from single to multiple islet autoantibodies. Our research will advance knowledge and establish a framework for improved T1D risk prediction and novel intervention strategies through targeting RBPs. RBP dysregulation has been implicated in multiple diseases and targeting RBPs is a feasible therapeutic approach. A potential outcome is the identification of novel gene isoforms and RBPs that can serve as therapeutic targets based on alternative splicing and regulation to intervene in the transition of islet autoimmunity in T1D.
Relevance to T1D
Type 1 diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-secreting beta cells by autoreactive T cells. In the general population, the risk of T1D is ~0.3%. However, the risk of T1D in a person with a first-degree relative with T1D increases to ~8%, highlighting the significant role genetic factors play in determining T1D risk. Environmental factors, though currently unknown, play a role equal in importance to genetic factors in the development of T1D, as shown by ~50% of identical twins (who have the same DNA sequences) differ in disease status. We have pioneered the discovery of over 100 regions in the human genome that contain genetic variation associated with T1D risk. The majority of these alterations in DNA are predicted to regulate expression of genes in immune system cells (such as CD4+ T cells).
While the initial trigger of the autoimmune attack on the beta cells that leads to T1D is unknown, the autoimmune attack can be detected through the presence of one or more pancreatic islet autoantibodies in the blood, specifically IAA, IA-2A, GADA, and ZnT8. The presence of a single islet autoantibody increases the risk of T1D to 10%-15%; in contrast, the development of a second islet autoantibody raises the T1D risk to over 70% (now considered stage 1 T1D), with most individuals eventually progressing to reduced glucose tolerance (stage 2 T1D) and, ultimately, clinical (stage 3) T1D. Only a portion of those individuals with a single islet autoantibody progress to multiple islet autoantibodies, and the factors that characterize this important transition point are unknown. The biological factors that are critical to this transition from single to multiple islet autoantibodies will provide knowledge for improved screening, monitoring, and novel targets for intervention to prevent this transition.
As regulation of gene expression is an important biological mechanism associated with differences between individuals with T1D and those without T1D, we have evidence that ‘alternative splicing’ may play an important role in T1D risk. Alternative splicing is a process by which a single gene, depending on environmental cues, combines specific sequences that code for proteins. This rearranging of coding sequences allows cells to respond to environmental cues and produce multiple proteins with slightly different functions. Alternative splicing events are tightly regulated by the action of RNA-binding proteins (RBPs). We propose to determine how alternative splicing and impaired RBP function can lead to failed immune regulation in CD4+ T cells and impact the transition from a single to multiple islet autoantibodies. Impaired RBP regulation has been implicated in cancer and other diseases, and therapies that target RBPs is feasible through modern molecular approaches, including an FDA-approved use of antisense oligonucleotides (ASOs) for the treatment of spinal muscular atrophy. This ASO modulates RBP-mediated splicing and serves as a proof-of-concept for targeting splicing regulation to intervene, an approach that has relevance to disrupt the progression from single to multiple islet autoantibodies and development of T1D.