The effect of diabetes and the common diabetogenic TBC1D4 p.Arg684Ter variant on cardiovascular risk in Inuit in Greenland

Cardiovascular disease (CVD) is a well-known complication of diabetes, but the association has not been studied among Inuit in Greenland. The aim was to examine the association between diabetes and incident CVD among Inuit in Greenland and determine if the common diabetogenic TBC1D4 variant confers increased risk of CVD. We followed an initial study population of 4127 adults in Greenland who had participated in at least one population-based health survey, in national registers. We used Poisson regression to calculate incidence rate ratios (IRR) of cardiovascular endpoints, comparing participants with and without diabetes and comparing homozygous TBC1D4 carriers with heterozygous carriers and non-carriers combined. Close to 10% had diabetes and age range was 18–96 years (45% male). Of the 3924 participants without prior CVD, 362 (~ 9%) had CVD events during a median follow-up of 10 years. Multivariate IRR for the effect of diabetes on CVD was 1.12 (95% CI: 0.80, 1.57) p = 0.50. Using a recessive genetic model, we compared homozygous TBC1D4 carriers with wildtype and heterozygous carriers combined, with a multivariate IRR of 1.20 (95% CI: 0.69, 2.11) p = 0.52. Neither diabetes nor the TBC1D4 variant significantly increased CVD risk among Inuit in Greenland in adjusted models.

Scientific Reports | (2020) 10:22081 | https://doi.org/10.1038/s41598-020-79132-1 www.nature.com/scientificreports/ of 0.06% in 1962 3 in contrast to recent estimates of diabetes of 9% and prediabetes of 19% 4 . Diabetes increases risk of CVD two to three fold in European populations 5 and the same trend is shown in Inuit populations 6,7 including Inuit in Greenland 8,9 . However, no epidemiological studies have been conducted to this day, elucidating the effect of diabetes on CVD risk in Greenland. Physical inactivity, obesity, ageing, poor diet, smoking, abnormal lipids, hypertension and genetic predisposition are common risk factors for diabetes [10][11][12] and CVD [13][14][15][16][17] . More than half of the population in Greenland smoke 18 and physical inactivity is much more common in modern days as a result of a social transition from a traditional active lifestyle to a modern sedentary life with access to processed high caloric foods 19 . Accordingly obesity is an increasing problem among Inuit in Greenland with a rise from 15 to 25% over the past two centuries 20 and it is a major risk factor of diabetes 21 . The common myth that Inuit are exempted from CVD [22][23][24] is continuously disproven with recent studies finding that CVDs in Inuit populations are prevalent and relevant public health issues [25][26][27] . Adding to the pool of risk factors for diabetes among Inuit is the unique genetic structure of isolated populations giving rise to common risk alleles with larger effect sizes than in populations with a more diverse genetic composition.
The TBC1D4 p.Arg684Ter is one such risk variant discovered in the Greenlandic population in 2014 with an allele frequency of 17% and 4% homozygous (HO) carriers 28 . The diabetogenic effects demonstrated include decreased insulin-stimulated glucose uptake in skeletal muscle leading to postprandial hyperglycemia and hyperinsulinemia, impaired glucose tolerance and diabetes 28 . The variant shows the strongest association using a recessive model, and HO carriers have an odds ratio of 10.3 for developing diabetes and the variant is thought to explain around 15% of all diabetes in Greenland 28 . Among Inuit in Nunavik, a population comparable to Inuit in Greenland, diabetes was found to be underdiagnosed among carriers of the variant 29 , underlining the importance of recognizing potential clinical implications associated with the TBC1D4 variant.
The pathophysiology connecting the TBC1D4 variant to diabetes involves the defective protein it produces. The long isoform of TBC1D4 expressed in skeletal and heart muscle codes for an insulin stimulated activating protein that facilitates GLUT4 translocation from intracellular vesicles to the cell membrane after insulin stimulation 30 . Lack of the long TBC1D4 isoform confers postprandial hyperglycemia and hyperinsulinemia. A potential link between TBC1D4 and CVD is supported by mice knock-out studies that have uncovered that TBC1D4 phosphorylation plays an important role in the electrical conduction system of the heart 31 suggesting that the variant may influence cardiovascular outcomes. Furthermore, a recent TBC1D4 study found that knock-out mice exhibited impaired cardiac function, increased infarction area and decreased left ventricular wall thickness three weeks after induced cardiac ischemia, compared to wild type (WT), indicating a functional role of TBC1D4 variant regarding heart damage after myocardial infarction 32 . A common ground between the TBC1D4 variant, diabetes and cardiovascular outcomes, is found in the "common soil hypothesis", a shared pathophysiological pathway of diabetes and CVD through obesity, insulin resistance, dyslipidemia and inflammation 33,34 .
We do not know if CVD risk for Inuit with diabetes is the same as for Caucasians with diabetes or if the TBC1D4 variant confers increased risk of CVD. The aim of the study was therefore to determine if diabetes is associated with CVD risk among Inuit in Greenland. The second objective was to examine if carriers of the TBC1D4 variant have a higher risk of CVD than non-carriers.

Methods
Study population. The study population included participants from two countrywide Greenlandic population-based studies: the B99 study and the Inuit Health in Transition (IHIT) study. B99 is a population-based survey of life style and disease among Greenlanders conducted from 1999-2001 and IHIT, completed from 2005-2010, is a general health study among adults in Greenland 35,36 . Participants over 18 years who completed an interview, a clinical examination and had blood samples drawn, were included in the study population. Participants who appeared in both surveys were included at the earliest participation date to get maximum follow up time. The population surveys are described in detail elsewhere 35,36 . The studies were approved by the Ethics Committee in Greenland with adherence to the Declaration of Helsinki and written informed consent was obtained from participants.

Definition of diabetes.
Diabetes was defined according to World Health Organization 2006 criteria for type 2 diabetes: fasting plasma glucose > 6.9 mmol/l or plasma glucose > 11 mmol/l 2 h after an oral glucose load during an Oral Glucose Tolerance Test (OGTT) and/or self reported by questionnaire 37 .
TBC1D4 genotyping. Participants were genotyped for the TBC1D4 variant using the KasPAR assay (LGC Genomics, Hoddesdon, UK) and admixture proportions of European ancestral DNA were estimated for each individual, where a proportion of 1 indicates 100% Inuit ancestry and zero indicates 100% European ancestry using data generated from the Illumina MetaboChip 28 . The Greenlandic Hospital Discharge Register. Since 1987, the Greenlandic Hospital Discharge Register has been used to register discharge diagnoses from health centers and hospitals in Greenland. Medical information and information on surgical procedures are recorded. In 2014 Greenland changed to a new electronic medical record system from AEskulap to Cosmic. Cosmic was first implemented at Queen Ingrid's Hospital in Nuuk in the beginning of 2014 and then successively among the 4 main regional hospitals and health centers throughout Greenland.
The Danish National Hospital Register. The Danish National Hospital Register was established in 1977 with the purpose of registering discharge diagnoses from Danish hospitals 43 . Greenlandic patients requiring specialized medical procedures or treatments unavailable in Greenland are flown to Copenhagen for treatment and their diagnoses are therefore found in this register.

Statistical analyses.
We examined the association of diabetes with CVD risk and the effect of the TBC1D4 p.Arg684Ter variant on CVD risk. Date of entry into the study was defined as date of the clinical examination during the health survey and participants were followed from entry until first CVD event, death, emigration or end of follow-up, whichever came first. CVD incidence rates were estimated using Poisson regression with log-person-time as offset variable and a significance level at 5%. Since CVD rates are not constant over time, the LEXIS macro was used to cut follow-up time into 1-year age bands and current age and calendar year were used as time scales 45 . Poisson regression was run on complete cases and participants with CVD events prior to entry were excluded from analyses. The effect of diabetes on CVD risk was estimated in a crude analysis and then adjusting for age, calendar year and sex in model 1. In model 2 we additionally adjusted for body mass index (BMI), systolic and diastolic blood pressure, low density lipoprotein (LDL) cholesterol, triacylglycerol (TAG) levels and smoking. In model 3 we further adjusted for the effect of TBC1D4 variant and admixture. The effect of TBC1D4 was estimated using a recessive model (the model with the best fit 28 ) comparing HO with wildtype (WT) and heterozygous (HT) combined, in a crude model and adjusting for the same confounders as in model 3. As CVD outcomes related to diabetes are not consistently defined in the literature, we did a sensitivity analysis excluding atrial fibrillation and heart failure and ran analyses again (CVD outcomes then only included ischemic heart disease, stroke and vascular disease).
To avoid exclusion of individuals with missing data on OGTT and TBC1D4 genotyping covariates, which may infer biased results 46 , we ran Multivariate Imputation by Chained Equation 47 with missing-at-random assumptions. For both OGTT and TBC1D4 missing data, we independently assessed five copies of the data, each with imputed missing values and estimates of parameters were averaged across the copies according to Rubin rules 48 . For imputation, we used R statistical program version 3.6.0 and the R-package mice 49 . Data management and Poisson regression was done using SAS Statistical Software (version 9.4) 50 .

Results
The baseline study population included 4127 participants who had completed lifestyle questionnaires and clinical examinations in the two health surveys B99 (n = 1317) and IHIT (n = 2810). Age range was 18 to 96 years and 1837 (44%) were male. We found 367 (~ 10%) participants with diabetes out of 3653 who had complete OGTT information (Table 1) and as such 474 participants had missing data on fasting and/or 120 min blood glucose values of the OGTT. Participants with diabetes had higher median age, BMI, systolic and diastolic blood pressure and fewer were smokers than in the normoglycemia group. HO carriers of the TBC1D4 variant were in higher number in the diabetes group compared to the normoglycemia group, n = 47 (13.7%) and n = 71 (2.3%) and conversely TBC1D4 WT genotype was lower in the diabetes group than the normoglycemia group n = 205 (59.8%) and n = 2205 (70.3%) respectively. Cardiovascular outcomes. We followed the initial study population of 4127 individuals in national registers for a median of 10.2 years interquartile range (IQR) [8.3, 13.8] until CVD event, death, emigration or end of follow-up (December 31st 2016) whichever came first. A total of 622 participants died during follow-up and 111 were CVD registered deaths. We excluded 203 individuals who had a CVD event registered prior to inclusion and of the 3924 individuals that remained 362 (9.2%) had incident CVD events (Table 2). We divided participants with complete OGTT data n = 3460 (464 with missing OGTT data) into diabetes (n = 319) and normoglycemia (n = 3141) groups that accounted for 60 (18.8%) and 283 (9.0%) CVD events respectively. In total, CVD outcomes were most commonly stroke (35.1%) and ischemic heart disease (34.8%) and to a lesser extent

Discussion
In this study we tested if diabetes and the TBC1D4 variant were associated with CVD risk in Inuit in Greenland. We found that diabetes was associated with elevated CVD risk in a crude analysis, however, in adjusted models the effect was not maintained. Assuming a recessive effect the TBC1D4 variant showed a maintained trend of increased CVD risk, however this trend was not significant and a protective effect on all-cause mortality was not significant either. Age, male sex and LDL cholesterol significantly increased CVD risk in adjusted models as has been described in Inuit populations before 51 . In total 9% of normoglycemic participants and 19% of participants with diabetes developed incident CVD events. Altogether ~ 10% of participants had diabetes, as has been described before in the Greenlandic population 52 . Diabetes conferred a significant increase of all-cause mortality in a multi-adjusted model. It was unexpected that diabetes did not confer statistically significant increased CVD risk, as is the case in many other non-Inuit populations 5,53-55 and in Inuit in Alaska 51 . We propose several reasons to explaining this. One reason could be if CVD outcomes were not defined accurately. The literature is rich in studies supporting that diabetes increases CVD risk, but definition of CVD outcomes varies greatly. Agreeing upon which CVD diagnoses are diabetes relevant is difficult and especially atrial fibrillation and heart failure may cause discussions among physicians. We chose to include atrial fibrillation 23,56,57 and heart failure [58][59][60] , supported by the literature as relevant complications to diabetes, in addition to stroke, ischemic heart disease and vascular disease. In a sensitivity analysis we omitted atrial fibrillation and heart failure. Age, male sex and LDL remained significant and in addition we found that smoking, calendar year and European admixture significantly increased risk of CVD. The effect of smoking could be explained by its pathophysiological mechanisms causing atherosclerosis, which is a dominant factor in the pathophysiology of especially ischemic heart disease, vascular disease and stroke 51 . Atrial fibrillation and heart failure have multiple other pathophysiological mechanisms besides atherosclerosis harmonizing well with the effect of smoking found when these were omitted. The elevated CVD risk per increase in calendar year may walk hand in hand with the traditional Inuit diet of marine animals steadily over time being replaced by imported foods of low quality, thus increasing CVD associated risk 62 . One study of Inuit in Alaska found that a traditional Inuit diet was associated with lower TAG, blood pressure and slightly higher LDL, concluding that it was linked to a better cardiovascular profile 63 . The fact that Inuit admixture had a protective effect on CVD risk in the sensitivity analysis, could also be related to the Inuit diet being more habitual among inhabitants of smaller more isolated villages in Greenland, where Inuit admixture levels are highest 64 .
A second reason could be that Inuit are not directly comparable to Caucasians due to genetic differences. Genes play a powerful role in isolated populations as seen among Pima Indians where close to half of the population is diagnosed with diabetes 65,66 . Similarly, the TBC1D4 variant convincingly increases diabetes risk in Inuit populations, but it does not seem to convincingly increase CVD risk. An explanation could be, that there could be undiscovered CVD protective variants or Inuit specific interactions with diet or metabolic traits affecting CVD risk, or the TBC1D4 variant could induce less dangerous diabetes form like GCK-MODY (Glycokinase Maturity Onset Diabetes of the Young). GCK-MODY patients have prolonged mild hyperglycemia but do not require treatment 67 . The TBC1D4 variant showed a protective but not statistically significant effect on all-cause mortality and diabetes significantly increased risk of all-cause mortality, supporting a theory that TBC1D4 diabetes may have fewer clinical implications than non-TBC1D4 diabetes.
A third reason could be that diabetes duration was not long enough to confer increased CVD risk, as diabetes incidence in Greenland has increased from virtually non-existent to ~ 10% over the last half a century. Perhaps in 50 years, diabetes associated CVD risk will be comparable to Caucasian populations. CVD risk increases with age and although life expectancy in Greenland has gone up from 63 to 72 years, compared to the mean of the European Union of 72 to 82 years over the past 40 years 68 , it remains substantially lower and a younger population will develop fewer CVD events.
A fourth reason could be changes in health behavior after receiving a diabetes diagnosis. Participants with diabetes in this study were screen detected in population-based studies and were identified early in time, possibly before any diabetes symptoms manifested themselves. Consequently, participants identified with diabetes would have had time to change their risk profile by e.g. smoking secession, weight loss, a healthier diet and so on. We found fewer smokers in the diabetes group compared to the normoglycemia group 54.8 vs 69.4%. As such CVD risk may be underestimated.
A fifth explanation could be an imprecise definition of diabetes in this population. We used self-reported and OGTT (fasting and two-hour glucose measurements) to define diabetes, as WHO guidelines suggested at the time the health surveys were conducted. Using glycated hemoglobin (Hba1c) we would have caught an overlapping but not identical group of people with diabetes. A study with Inuit in Canada with the same allele frequency and postprandial effect of TBC1D4, found that in carriers with prediabetes or type 2 diabetes, 32% would have remained undiagnosed without an OGTT 29 . Defining diabetes by Hba1c instead of OGTT criteria as been shown to affect ethnic groups differently 69 . Inuit have, for any given glucose value by OGTT, a higher Hba1c compared to Danes, suggesting different associations between Hba1c and blood glucose levels in these populations 70 .
A sixth reason could be if the registers with CVD outcomes were incomplete or faulty. However this seems unlikely, because two studies validated the Greenlandic Hospital Discharge Register in its entirety 71 and CVD diagnoses separately 72 and both found it valid for epidemiological purposes. Diagnoses in the Danish Hospital Discharge Register have also been validated with good results 43 .
Regarding the effect of the TBC1D4 variant, we found maintained increased but statistically insignificant risk of CVD for HO carriers. We handled missing genotypes by redoing Poisson regression after multiple imputation and estimates were not changed significantly, suggesting that the number of complete cases in the models are representative of the full sample. www.nature.com/scientificreports/ Compared to non-carriers, HO carriers had more frequent ischemic heart disease (HO 46.7% vs WT 32.7%), CVD deaths (33.3% vs 27.2) and fewer stroke diagnoses (HO 20.0% vs WT 35.5%), corresponding well to the fact that TBC1D4 is expressed in heart and muscle tissue and not in the brain. Although we could not detect a significantly increased risk of CVD for the TBC1D4 variant, we cannot completely rule one out. Since the TBC1D4 variant is present with the same allele frequency among Inuit in Canada 29 , a way to get more power could be to pool data from Greenland and Canada. If there is an increased risk of CVD for HO carriers of the TBC1D4 variant, it would be of public health interest to uncover it.

Conclusions
In conclusion, this study showed that neither diabetes nor the TBC1D4 variant significantly increased CVD risk in Inuit in Greenland. However, we find it imprudent to draw hasty conclusions and suggest more studies be conducted on diabetes and CVD risk in Inuit populations.
Ethics approval and consent to participate. Oral and written consent was obtained from all participants in the two health surveys (B99 and IHIT) and the committee for Research Ethics in Greenland (Commission for Scientific Research in Greenland) granted ethical approval. The study protocols were in accordance with the Helsinki Declaration.

Data availability
Data of the health surveys is available upon request for relevant purposes.