The broad pathogenetic role of TCF7L2 in human diseases beyond type 2 diabetes

Abstract The TCF7L2 protein is a key transcriptional effector of the Wnt/β‐catenin signaling pathway, regulating gene expression. It was initially identified in cancer research and embryologic developmental studies. Later, the TCF7L2 gene was linked to type 2 diabetes (T2D), implicating TCF7L2 and Wnt‐signaling in metabolic disorders and homeostasis. In fact, TCF7L2‐T2D variants confer the greatest relative risk for T2D, unquestionably predicting conversion to T2D in individuals with impaired glucose tolerance. We aim to describe the relevance of TCF7L2 in other human disorders. The TCF7L2‐single nucleotide polymorphisms (SNPs) and T2D‐risk association have been replicated in numerous follow‐up studies, and research has now been performed in several other diseases. In this article, we discuss common TCF7L2‐T2D variants within the framework of their association with human diseases. The TCF7L2 functional regions need to be further investigated because the molecular and cellular mechanisms through which TCF7L2 contributes to risk associations with different diseases are still not fully elucidated. In this review, we show the association of common TCF7L2‐T2D variants with many types of diseases. However, the role of rare genetic variations in the TCF7L2 gene in distinct diseases and ethnic groups has not been explored, and understanding their impact on specific phenotypes will be of clinical relevance. This offers an excellent opportunity to gain a clearer picture of the role that the TCF7L2 gene plays in the pathophysiology of human diseases. The potential pleiotropic role of TCF7L2 may underlie a possible pathway for comorbidity in human disorders.

The association of TCF7L2 with T2D is one of the most powerful genetically discoveries in studies of complex diseases, as it has been consistently replicated in multiple populations with diverse genetic origins (Del Bosque-Plata et al., 2021). TCF7L2 and other proteins of the Wnt network have been related to various human disorders, reflecting their role in their pathogenesis (Lucero et al., 2010).
This review describes what is known to date about the TCF7L2 T2D-variants association in human diseases other than T2D.

| Statistical measures
We report ORs and p values (p) related to the data. The OR is a measure of association between an exposure (or a genetic variant) and an outcome (or a disease). The OR represents the odds that a disease will occur given a particular gene variant under study, compared to the odds of the disease occurring in the absence of that genetic variant. For OR = 1, the variant does not affect odds of disease; for OR > 1, the variant is associated with higher odds of disease; and for OR < 1, the variant is associated with lower odds of disease. P is the level of significance of a statistical test, representing the probability of the occurrence of a given event. In all analyses, a p < 0.05 was considered statistically significant, meaning that the probability that the data are due to random choice is <.05.
The p(trend) reflects the trend) of p, thus representing the probability that the data reflect a random result and not a true association.
2 | TCF7L2-T2D VARIANTS AND MISSION, BEYOND T2D T2D is the most common form of diabetes, characterized by increased glycemia contributed by impaired insulin action or insulinresistance (IR), and beta-cell dysfunction, and it is associated with obesity (Carlsson, 2019).
In Table 1, we show the role of the T2D-risk variants studied in diverse pathologies (Table 1), and in Table 2, we show the most common TCF7L2 variants with pleiotropic effects in human diseases (Table 2).

| Type 1 diabetes (T1D)
T1D, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin, a hormone that is necessary for survival because it allows glucose to enter cells to produce energy (WHO, 2020 These results suggest that, in children with only mild islet autoimmunity, mechanisms associated with TCF7L2 genetic variation contribute to the development of T1D, and this contribution is greater in the absence of obesity. It is plausible to think that the rs7903146-TT variant is most deleterious in the perinatal stage, thus reducing in carriers the chance of surviving pregnancy, childbirth, and lactation (Redondo et al., 2014).
To know if TCF7L2 locus influences progression of T1D-islet autoimmunity, Redondo et al. evaluated in relatives of T1D-patients single to multiple (≥2) autoantibody positivity .
DEL BOSQUE-PLATA ET AL.
Specifically, TCF7L2-T2D risk allele rs4506565, in linkage disequilibrium (LD) with T2D-variant rs7903146, reduces the risk of progression to multiple autoantibody positivity among single GAD65 autoantibody-positive subjects, particularly if T1D-risk HLA-haplotypes are absent. In subjects with single microinsulin autoantibody (mIAA) or IA-2 autoantibody, the rs7903146-T risk allele interacts with obesity or overweight status to mark faster progression to multiple autoantibody positivity. Thus, the T2D-associated TCF7L2 locus influences progression of islet autoimmunity, with differential effects given by autoantibody specificity and interaction of obesity or overweight status. The findings suggest a role of the TCF7L2-locus in islet autoimmunity in subsets of individuals and support an interrelationship between genetic, immunologic, and metabolic factors in T1D pathogenesis .
Larger studies are required to define whether the genetic sensitivity of islet β-cells to immunologic or metabolic stress contributes to the risk of developing diabetes, irrespective of its type (Leslie & Grant, 2018).

| Gestational diabetes mellitus (GDM)
GDM is defined as any degree of glucose intolerance that starts, or that is first recognized, during pregnancy. The definition applies whether insulin or only diet modification is used for treatment and whether or not the condition persists after pregnancy (Franz et al., 1994).
TCF7L2 is associated with susceptibility to GDM independently of the T1D-risk HLA-DQB1*0602 allele, autoantibodies to pancreatic β-cells, and other factors (e.g., maternal age, number of pregnancies, familial history of diabetes, and other genotypes).
In 168 pregnant Caucasian women, the rs7903146-TT genotype was associated with a more than fivefold increased GDM-risk (OR = 5.4; 95% CI = 1.5-19.3) (Franzago et al., 2017 (Noble et al., 1996). Although T2D-genetic risk is attributed to many common genetic variants contributing a small amount to T2D heritability (Fuchsberger et al., 2016), LADA-patients carry an increased TT-genotype frequency at the TCF7L2-T2D rs7903146 variant compared to controls. (15% vs. 6%; p = 0.03) (Cervin et al., 2008;Szepietowska et al., 2010). A meta-analysis demonstrated that the rs7903146-T variant is a population-independent susceptibility locus for LADA in Europeans. The effect size is similar for LADA and T2D. The genetic effect on the risk for LADA may be modulated by BMI; the lower the BMI, the greater the genetic effect (Lukacs et al., 2012). In LADA patients, TCF7L2-T2D common variants rs12255372 and rs7903146 are associated only with low Glutamic Acid Decarboxylase Antibody (GADA) titers, showing heterogeneous genetic and clinical characteristics in LADA patients according to GADA titers; the authors hypothesize that the TCF7L2-risk alleles predispose to lower insulin secretion in these patients (Zampetti et al., 2010).

| OBESITY
Overweight conditions and obesity are defined as abnormal or excessive fat accumulation that presents a risk to health. A BMI ≥ 25 is considered overweight, and ≥ 30 is obese (WHO, 2020). Obesity is determined by a complex interaction of environmental factors with genetic variants implicated in pathways of glucose-homeostasis regulation, thereby potentially modulating T2D development in obese subjects (Kahn, 2003;Mokdad et al., 2003). Obese carriers of the T2D compared to those with other genotypes, and this relationship appears even stronger after adjusting for obesity-onset age, male gender, dyslipidemia, and lower serum total adiponectin (Wrzosek et al., 2019). In addition, the risk of developing impaired fasting glycemia associated with the TT-genotype is stronger in obese than nonobese Caucasians (Yan et al., 2010).
Significantly, the rs7903146-T variant has been associated with gastric functional alterations potentially predisposing to obesity (e.g., reduction of fasting gastric volume and rapid gastric emptying) (Vazquez-Roque et al., 2011). In contrast, rs7930146-T has been associated with T2D in obese Polish adults.
Furthermore, a study in Mexican children showed that the rs12255372-T variant protects them from obesity, although replication is needed (Klünder-Klünder et al., 2011). Vascular GLP-1 receptor, a target of T2D-therapy, and TCF7L2 expression were significantly downregulated in obese subjects, indicating that TCF7L2 modulates vascular GLP-1 receptor expression (Kimura et al., 2018).

| METABOLIC SYNDROME (MetS)
MetS is a heterogeneous disorder defined by the presence of at least three of the following traits: waist circumference >40 inches in men or >35 inches in women, fasting glycemia >100 mg/dl or therapy, blood pressure >130 mmHg systolic or 85 mmHg diastolic or therapy, and triglyceridemia >150 mg/dl or therapy, high-density lipoprotein <40 mg/dl in men or <50 mg/dl in women or therapy (Grundy et al., 2005). TCF7L2 rs7903146-T influences MetS-risk, which increases with female gender and saturated fatty-acids intake (Phillips et al., 2012).
Also, rs7903146-T and rs12255372-T variants play a key role in

| DIABETIC NEPHROPATHY (DN)
Diabetic nephropathy, also known as diabetic kidney disease (Kittell, 2012), is the chronic loss of kidney function occurring in those with diabetes mellitus. DN is one of the leading causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD) (Longo et al., 2012). It has been classically defined by the presence of proteinuria >0.5 g/24 h (Mogensen, 1984 (Gross et al., 2005).
The role of TCF7L2 in CKD progression has been tested in several population-based studies, including the Atherosclerosis Risk in Communities Study (ARIC; N = 11,061 self-identified whites and N = 4,014 blacks). The T2D-risk alleles at rs7903146 and rs7901695 were significantly associated with CKD progression among ARIC participants and those without baseline T2D (Köttgen et al., 2008). In American Indians with early-onset DN, TCF7L2 variants have been associated with lower estimated glomerular filtration rate, but not with urinary albumin-to-creatinine ratio (Franceschini et al., 2012).
In 1 In the prospective ARIC-cohort study (13,117 middle-aged adults, cancer-free in the years 1987-1989), the rs7903146-T allele was analyzed in relation to various common cancers (Folsom et al., 2008). In 2000, T2D was found to have an inverse marginal incidence with prostate cancer; no association was found between T2D and the incidence of colorectal cancer (CRC), CC, cancer of the lung, or of the breast (Folsom et al., 2008). The rs7903146-T allele is associated with lung cancer in Caucasians but not in African-Americans. Furthermore, the rs7903146-T allele, presenting with a frequency of 30% in Caucasians, is associated with the risk of CC in the general population (approximately 17%). This association with lung cancer appears due to an independent genetic effect that is not explained by the presence of T2D (Folsom et al., 2008).

| CRC and CC
German T2D-patients carrying the rs7903146-T allele had an increased risk of CRC (p(trend) = 0.02). Further analysis revealed gender-specific effects: the rs7903146-T allele was associated with an increased risk of CRC only in women (p(trend) = 0.003) (Sainz et al., 2012). β-Catenin accumulation, and its mediated transactivation of certain TCF7L2-target genes, plays a key role in colorectal carcinogenesis (Shitashige et al., 2007). Another study did not confirm rs7903146-T association with CC (1578 cases, 1966 controls), but did find a statistical interaction between the variant and the recent use of aspirin and/or nonsteroidal antiinflammatory drugs (NSAIDs), suggesting that CC risk associated with rs7903146-T is mediated by the use of aspirin and/or NSAIDs (Freedman et al., 2009). There is evidence that CC predisposition is associated with an enhancer element containing the TCF7L2-rs6983267 variant, whose homozygosity increases CC risk circa 1.5 times, and the G allele increases TCF7L2 transcription factor binding in vivo and in vitro.
Complete genomic chromatin immunoprecipitation (ChIP) assay reveals this element as the strongest TCF7L2-binding site (Tuupanen et al., 2009). By a genomic binding profile using the CC cell line LS174T, it was identified that TCF7L2 binds to 6,868 genomic sites. In the human intestine, the majority of TCF7L2-transcriptional targets present on the same TCF7L2-DNA molecule (called "target in cys"), and not in a DNA molecule on which TCF7L2 is acting (called "targets in trans"), have been defined by combining genetic information with expression data. The motif-search algorithm defines the TCF7L2-binding site as an evolutionconserved AC/GA/TTCAAAG motif, although 30% of the binding regions do not contain a typical binding motif, which could be due to factors such as the presence of nonidentified atypical motifs, protein-protein interactions with other factors directly recruited to those regions, or to the fact that some TCF7L2-association signals reflect association at other sites (Hatzis et al., 2008).
By using the CRC line HCT116, ChIP, and sequencing, a total of 1,095 discrete sites binding to TCF7L2 were found in the genome, and a subgroup of 548 sites was found within 5 kb of sequences annotated in the National Center for Biotechnology Information Reference Sequences. Analysis of the metabolic pathways involved in these binding sites demonstrated that the most representative functions were related to T2D, coronary artery disease, and other significant disease risk loci, of which most were primarily represented by metabolic and cardiovascular traits, and a few were related to cancer and inflammatory and CVDs. The most enriched specific traits were T2D and height (Kasper et al., 2009;Zhao et al., 2010).

| Prostate cancer
Various studies and meta-analyses report that patients with T2D have a lower risk of developing prostate cancer. Even if the concentration of sex hormones and the expression of sex hormone receptors are among the main risk factors for a higher incidence of prostate cancer, carrying the TCF7L2 rs12255372-T allele is associated with prostate cancer progression and metastases. Also, studies show that the risk of death from prostate cancer increased in subjects with T2D compared to those without T2D (Kasper et al., 2009;Lee et al., 2016;Lucero et al., 2010;Meyer et al., 2010).

| Breast cancer
A study with 387 patients with breast cancer compared to healthy subjects found that the rs7903146-T allele was significantly associated with lymph node involvement (p = 0.003), while the rs12255372-T allele was not associated with clinicopathological characteristics (Naidu et al., 2012).

| PSYCHIATRIC DISORDERS
A psychiatric disorder is a mental illness that greatly disturbs an individual's thinking, moods, and/or behavior and seriously increases the risk of disability, pain, death, or loss of freedom.

| Schizophrenia (SCZ)
SCZ is characterized by distortions in thinking, perception, emotions, language, sense of self, and behavior. Common experiences include hallucinations (hearing voices or seeing things that are not there) and delusions (fixed, false beliefs) (WHO, 2020).
Exposure to antipsychotics induces metabolic dysregulation in some patients; however, SCZ is per se associated with increased T2D-risk in drug-naïve patients (Fernandez-Egea et al., 2009). In African-American patients with psycho-affective or schizophrenic disorders, the TCF7L2 rs7903146-T variant was associated with T2D (OR = 1.4; p = 0.03) by the additive model, and more so by the recessive model (OR = 2.4; p = 0.004).
Of interest, marginal significance was found for interaction between rs7903146-T and antipsychotic treatment (Irvin et al., 2009).
Alkelali et al. reported linkage of the 10q24-q26 region to SCZ in Arab-Israeli families with multiple SCZ-affected individuals (Alkelai et al., 2012). Within the identified locus, they found significant SCZ association with the TCF7L2-rs12573128 intronic SNP (p = 7.01 × 10⁻ 6 ) and the nearby rs1033772-intergenic SNP (p = 6.59 × 10⁻ 6 ). These results independently support previous findings regarding a possible role of TCF7L2 in SCZ susceptibility (Alkelai et al., 2012). In the Danish population, the rs7903146-T allele was associated with SCZ in the discovery sample (p = 0.0052) and in the replication sample (OR = 1.07; 95% CI = 1.01-1.14; p = 0.033) (Hansen et al., 2011). In 191 elderly T2Dpatients, four TCF7L2 SNPs (rs7901695, rs7903146, rs11196205, and rs12255372) were tested for association with volumes of white matter hyperintensities, gray matter, amygdala, and hippocampal imaging by using brain magnetic resonance. TCF7L2 polymorphisms were found to be associated with a lower volume of the amygdala. These findings should be confirmed in other populations and expanded in other regions of the limbic system. Carriers of these TCF7L2 polymorphisms may be predisposed to increased risk for schizoid disorders (Ganmore et al., 2019).

| Bipolar disorder (BD)
BD is a serious mental illness in which common emotions become intensely and often unpredictably magnified; individuals with BD can quickly swing from extremes of happiness, energy, and clarity to sadness, fatigue, and confusion (WHO, 2020).
BD is a complex disease associated with several hereditary traits, including a higher BMI. Although overall there was no evidence for

| CYSTIC FIBROSIS (CF)
CF is a chronic lung disease causing accumulation of thick and sticky mucus in the lungs, altering the pancreas and contributing to CF-secondary diabetes, and impairing other organs. In CF, the DEL BOSQUE-PLATA ET AL.

| ENDOCRINE DISORDERS
Endocrine disorders are diseases related to the hormone-producing glands of the body. These disorders can affect multiple parts of the body and have a wide range of signs and symptoms, depending upon the gland being affected (Kronenberg et al., 2016).

| Premature adrenarche (PA)
PA, also referred to as premature pubarche, refers to the early appearance of pubic hair, axillary hair, or both in children without other signs of puberty (Voutilainen & Jääskeläinen, 2015).
The rs7903146-T allele is more frequent in PA subjects with low height and weight than in controls, suggesting that this allele may be involved in the pathogenesis of PA in lean subjects (Lappalainen et al., 2009).

| Polycystic ovarian syndrome (PCOS)
PCOS, a common endocrine disorder characterized mainly by irregular menses and hyperandrogenism, is frequently associated with IR and obesity. The role of IR and obesity in this syndrome is not clear; thus, the role of obesity and T2D-variants has been investigated in PCOS and its specific traits (Rojas et al., 2014).
In an association study with 386 PCOS patients (defined by the Rotterdam criteria) and 1,971 control women, the rs7903146-T variant was strongly associated with body weight (Biyasheva et al., 2009

| CARDIOVASCULAR DISEASES (CVD)
CVDs are a group of disorders of the heart and blood vessels, including coronary heart disease and cerebrovascular disease. Four out of five CVD deaths are due to heart attacks and strokes, and a third of these deaths occur prematurely in people under 70 years of age (WHO, 2020).
The rs12255372-TT and rs7903146-TT genotypes are associated with reduced insulin sensitivity and high cardiosympathetic activity.
These effects are independent of GLP-1 and insulinemia, which suggests the potential role of these variants in increased cardiovascular risk through the augmentation of central nervous system (CNS)-sympathetic activity (Boccardi et al., 2010). It has also been reported that body mass modifies the association of the rs7903146-T allele with incidental coronary heart disease risk (Kucharska-Newton et al., 2010;Zhao et al., 2010).

| CONCLUSIONS
Although the role of the TCF7L2 T2D-variants has been extensively studied in a number of diseases, this has opened the flood-gates for an entirely new series of challenging questions. Previous findings suggest that genetic variation in different regions of the TCF7L2 gene is associated with distinct phenotypes, but the pathophysiological mechanisms-related pathways are not well known.
As ethnic variation in allele frequencies has been found throughout the genome, it is of great importance to study its genetic diversity in different races, ethnicities, and populations to reveal evolutionary patterns and identify variants that contribute to the causes of common diseases.
Within the Wnt pathway, TCFs function can be inhibitory and activating dependent on the context; more research is needed in this area. In particular, dissecting the TCF7L2 functional regions and understanding their impact on specific phenotypes will be important to comprehend their role in diverse human diseases and highlight a possible pleiotropic role in human comorbid disorders.

| PERSPECTIVES
Fifteen years ago, intronic variants in TCF7L2 were associated with T2D; since then, a huge amount of knowledge has been accumulated in diverse contexts, opening up enormous possibilities for follow-up.
Among the scientific opportunities to explore are the mechanisms over which TCF7L2 exerts its effect on diverse diseases, which may vary depending on the context of the specific cell or tissue. A deeper knowledge of the TCF7L2 variants and their potential differential cellor tissue-specific role along with a deeper knowledge of a more precise role of the gene variation in diverse races and ethnicities, as well as an understanding of the role of variation in natural selection, will lead us to a more thorough understanding of the gene and its variants effects in disease.
The Wnt/β-catenin signaling is subject to regulation at different levels, and its effects vary by cellular, temporal, and spatial contexts.
Thus, it is difficult to generalize whether any end pathway results in activation or inhibition. A clear example is within cancer, where, if oversimplified, the Wnt/β-catenin signaling and TCF7L2 pathways can be considered oncogenic or tumor-suppressive.
We propose that research must be carried out to understand the TCF7L2 molecular mechanisms controlling both localized and temporal expression of their signaling components in the specific cells and tissues affected in different diseases. It is expected that highthroughput methods will help to resolve many questions and provide essential knowledge regarding Wnt/β-catenin regulation and its perpetual crosstalk with other key signal transduction pathways.
Using the integration of genomic data with different types of "omics" will elucidate how disease variants in TCF7L2 interact with Wnt/βcatenin signaling, according to the genomic background of each individual. This will further our understanding of the pathogenic role of the pleiotropic variants.
In addition, to study the role of Wnt/β-catenin signaling and its interaction with TCF7L2 in different diseases, we need to address how the animal and in vitro models recapitulate what is seen in patients with regard to deregulation of Wnt/β-catenin signaling. These discoveries should be harmonized especially with information related to Wnt/β-catenin activation or inhibition in human diseases.