TBC1D4 Disruption Is Common Among the Inuit

Diabetes Care Volume 39, November 2016 1889 RCSO MEDICINE PRECISION

Despoina Manousaki,1,2 Jack W. Kent Jr.,3 Toward Precision Medicine: Karin Haack,3 Sirui Zhou,4,5 Pingxing Xie,4,6 Celia M. Greenwood,1,2,7 Paul Brassard,1,4 TBC1D4 Disruption Is Common Deborah E. Newman,3 Shelley Cole,3 Jason G. Umans,8,9 Guy Rouleau,2,6,10 Among the Inuit and Leads to Anthony G. Comuzzie,3 and Underdiagnosis of Type 2 Diabetes J. Brent Richards1,2,4,11 Diabetes Care 2016;39:1889–1895 | DOI: 10.2337/dc16-0769

OBJECTIVE A common nonsense mutation in TBC1D4 was recently found to substantially increase the odds of type 2 diabetes in Greenlandic Inuit, leading to exclusively increased 1Centre for Clinical Epidemiology, Department of postprandial glucose. We investigated the frequency and effect of the TBC1D4 mu- Epidemiology, Lady Davis Institute for Medical tation on glucose metabolism and type 2 diabetes diagnosis among Canadian and Research, Jewish General Hospital, McGill Uni- versity, Montreal, Quebec, Canada Alaskan Inuit. 2Department of Human Genetics, McGill Univer- sity, Montreal, Quebec, Canada RESEARCH DESIGN AND METHODS 3Texas Biomedical Research Institute, San Anto- Exome sequencing of the TBC1D4 variant was performed in 114 Inuit from Nunavik, nio, TX 4 Canada, and Sanger sequencing was undertaken in 1,027 Alaskan Inuit from the Department of Medicine, McGill University, Montreal, Quebec, Canada Genetics of Coronary Artery Disease in Alaskan Natives (GOCADAN) Study. Associa- 5Department of Medicine, UniversitedeMon-´ tion testing evaluated the effect of the TBC1D4 variant on diabetes-related metabolic treal,´ Montreal, Quebec, Canada traits and diagnosis. 6Montreal Neurological Institute and Hospital, Montreal, Quebec, Canada 7 RESULTS Department of Oncology, McGill University, Montreal, Quebec, Canada The TBC1D4 mutation was present in 27% of Canadian and Alaskan Inuit. It was 8MedStar Health Research Institute, Hyattsville, 2 strongly associated with higher glucose (effect size +3.3 mmol/L; P =2.5x10 6) MD and insulin (effect size +175 pmol/L; P = 0.04) 2 h after an oral glucose load in 9Georgetown-Howard Universities Center for homozygote carriers. TBC1D4 carriers with prediabetes and type 2 diabetes had an Clinical and Translational Science, Washington, DC increased risk of remaining undiagnosed unless postprandial glucose values were 10Department of Neurology and Neurosurgery, tested (odds ratio 5.4 [95% CI 2.5–12]) compared with noncarriers. Of carriers with McGill University, Montreal, Quebec, Canada 11 prediabetes or type 2 diabetes, 32% would remain undiagnosed without an oral Department of Twin Research and Genetic Ep- idemiology, King’s College London, London, U.K. glucose tolerance test (OGTT). Corresponding author: J. Brent Richards, brent. CONCLUSIONS [email protected]. Disruption of TBC1D4 is common among North American Inuit, resulting in exclu- Received 7 April 2016 and accepted 9 August 2016. sively elevated postprandial glucose. This leads to underdiagnosis of type 2 di- This article contains Supplementary Data online abetes, unless an OGTT is performed. Accounting for genetic factors in the care of at http://care.diabetesjournals.org/lookup/ Inuit with diabetes provides an opportunity to implement precision medicine in suppl/doi:10.2337/dc16-0769/-/DC1. this population. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not Precision medicine, as deﬁned by the National Institutes of Health’s Precision Medicine for proﬁt, and the work is not altered. More infor- Initiative Working Group, aims to tailor health care to individual variability in genes, mation is available at http://www.diabetesjournals environment, and lifestyles (1). While oncology has demonstrated some tangible suc- .org/content/license. cess with precision medicine, few examples exist in other domains of health care (2). See accompanying articles, pp. 1854, Genetic predisposition to disease may vary not only by individual but also by ancestry, 1858, 1870, 1874, 1879, 1896, 1902, and harnessing this information may help health care providers to better understand 1909, and 1915. 1890 Inuit TBC1D4 Variant and Type 2 Diabetes Diabetes Care Volume 39, November 2016

how to approach disease among different Alaskan Natives (GOCADAN) study (6) (the different ancestral populations with ethnicities. This may be particularly rele- Western North American Arctic). available data in phase 3 of the 1000 Ge- vant in specific populations, such as the Inuit from Nunavik were recruited nomes Project (8). Inuit, that have had different natural se- originally for the purposes of a research Next, we conducted a genetic associ- lection pressures leading to different fre- project assessing genetic determinants ation study of the GOCADAN cohort, ap- quencies of genetic variants. of fatty acid metabolism (7). While that plying both additive and recessive genetic Moltke et al. (3) recently identified a study did not include type 2 diabetes models to test for associations between population-specific nonsense variant in status or metabolic traits, it was used the TBC1D4 variant and reported type 2 TBC1D4 which was both common (minor to quantify the prevalence of the diabetes status, as well as 14 traits related allele frequency [MAF] 17%) and had a TBC1D4 variant in Nunavik. to type 2 diabetes (fasting and 2-h plasma large effect on type 2 diabetes risk among Informed consent for the current study glucose concentrations at OGTT, fasting the Greenlandic population (homozygote was obtained from all participants. The and 2-h serum insulin concentrations odds ratio [OR] 10.3; P =1.63 10224 in a TBC1D4 variant (rs61736969, a nonsense at OGTT, Gutt insulin sensitivity index recessive model). Specifically, compared polymorphism in TBC1D4 that is also [ISI0,120] (9), serum HbA1c, triglycerides, with noncarriers, homozygous carriers of called p.Arg684Ter or c.2050C.T) was total serum cholesterol, LDL cholesterol, the TBC1D4 p.Arg684.Ter variant had assayed using exome sequencing (see HDL cholesterol, albuminuria, BMI, percent- markedly higher concentrations of 2-h the Supplementary Data for a detailed age fat mass, and waist circumference). plasma glucose (3.8 mmol/L [68 mg/dL]; description of the exome sequencing). For these quantitative traits, the associa- P =2.53 10235) and serum insulin We next tested the effect of this variant tion testing was done only in participants (165 pmol/L [24 uIU/mL]; P =1.53 on glucose metabolism in the GOCADAN without diabetes. To evaluate the associa- 10220) after an oral glucose tolerance study (6). This longitudinal, population- tion between the TBC1D4 variant and test (OGTT). Conversely, this variant de- based study, conducted between October type 2 diabetes status, we used the full creased fasting glucose and had little effect 2000 and April 2004, aimed to investigate data set (both patients with diabetes and on HbA1c.Thesameeffectsdalthough of the genetic determinants of cardiovascu- those without diabetes). Because of skewed smaller magnitudedwere also observed lar disease among 1,214 Inuit from several values for some traits, we performed addi- in heterozygote carriers. coastal villages in the Norton Sound re- tional association tests after a rank-based What is not known is whether this var- gion of Western Alaska. All individuals inverse normal transformation (10) of the iant is present in other Inuit and whether older than 18 years residing in this re- values to ensure normality. The measured it may, given the exclusive effect on post- gion were invited to participate. Testing genotype association method used for the prandial glucose, influence diagnostic included a physical examination, labora- association testing was implemented in the strategies for type 2 diabetes. Such infor- tory determinations, and measures of Sequential Oligogenic Linkage Analysis Rou- mation may have clinical utility given that subclinical disease (described in detail tines software program (available from the prevalence of type 2 diabetes has elsewhere [6]). Specifically, type 2 diabe- https://www.nitrc.org/projects/se_linux; been increasing dramatically among the tes status was determined by self-report, described in detail elsewhere [11]). Finally, Inuit over the past 25 years (4). medication use, and fasting glucose we accounted for multiple testing by Interestingly, the ancestors of the Inuit screening (Accu-Chek Advantage). A urine applying a Bonferroni correction (12), divid- in Greenland are thought to have mi- sample was collected and blood samples ing the a level (0.05) by the number of traits grated from Siberia, across the Central were taken when fasted and 2 h after a tested (n = 14). We recognize that this cor- and Eastern Canadian Arctic (5). If the standard 75-g OGTT (Glutol; Paddock Lab- rection is overly conservative because many TBC1D4 nonsense mutation arose earlier oratory Inc., Minneapolis, MN). Partici- of the traits are highly correlated. along migration routes out of Siberia, this pants were excluded from the OGTT if To compare the risk of underdiagno- mutation would also be present in North they were already considered as having sis of prediabetes and type 2 diabetes American Inuit. Therefore, in this study type 2 diabetes according to the following among TBC1D4 carriers and noncarriers, we assessed the prevalence of the criteria: having known type 2 diabetes we applied the 2010 American Diabetes TBC1D4 mutation and its effect on diag- and requiring insulin, or using oral hypo- Association criteria (13) to the sample of nostic strategies for type 2 diabetes in the glycemic agents with two recorded glu- patients with available OGTT values in North American Arctic Inuit population. cose values .13.9 mmol/L or a fasting order to classify them into 3 groups: blood glucose value .12.5 mmol/L. type 2 diabetes, prediabetes, or neither. RESEARCH DESIGN AND METHODS For the purposes of this study, we used Specifically, type 2 diabetes was diag- To test whether the TBC1D4 variant is in- Sanger sequencing on the TBC1D4 variant nosed if at least one of the following deed specific to Greenland or is shared by in 1,027 GOCADAN participants (51 self- four criteria is present: 1)HbA1c $6.5% other Arctic populations, we assayed the reported cases with type 2 diabetes and (48 mmol/mol); 2) fasting plasma glucose p.Arg684Ter single nucleotide polymor- 976 controls) with available DNA samples. $7mmol/L(126mg/dL);3) 2-h plasma phism in two distinct North American cir- PCR and Sanger sequencing of the resulting glucose $11.1 mmol/L (200 mg/dL) dur- cumpolar populations. The first consisted amplicons were performed using estab- ing 75-g OGTT; or 4) random plasma of 114 Inuit from Nunavik in Northern Que- lished protocols (see the Supplementary glucose $11.1 mmol/L (200 mg/dL). Pre- bec, Canada (the Eastern North American Data for a detailed description of the diabetes was diagnosed if at least one of Arctic), and the second of 1,027 individuals method). the following three criteria was present: 1) from a cohort study of Alaskan Inuit, called For comparison purposes, we que- fastingplasmaglucosebetween5.6and the Genetics of Coronary Artery Disease in ried the MAFs of the TBC1D4 variant in 6.9 mmol/L (100 and 125 mg/dL); 2) care.diabetesjournals.org Manousaki and Associates 1891

2-h plasma glucose in 75-g OGTT be- analysis because of high levels of admixture increased concentrations of 2-h plasma glu- tween7.8and11.0mmol/L(140and (see the Supplementary Data for a defini- cose (b = +3.3 mmol/L; P =2.53 1026)and 199 mg/dL); or 3) HbA1c between 5.7% tion of the admixture). The genotyping of 2-h serum insulin concentrations (b =+175 and 6.4% (39 and 46 mmol/mol). the C/T variant in the remaining 104 sam- pmol/L; P = 0.04) after an OGTT. Homozy- Then we compared the ratio of TBC1D4 ples from Nunavik revealed an allele fre- gous carriers also demonstrated decreased carriers to noncarriers who would have quency of 16.3% for the T allele (the peripheral insulin sensitivity, as estimated been diagnosed as having prediabetes nonsense polymorphism) (Supplementary by the ISI0,120 (b = 21.36 SDs; P =0.001). and type 2 diabetes only by a 2-h OGTT. Table 1). Heterozygous carriers without diabetes The OR of missed diagnosis of type 2 di- Sanger sequencing of the p.Arg684Ter also showed an increase in 2-h plasma glu- 2 abetes was calculated using the Fischer variant in the GOCADAN cohort also re- cose (b = +0.5 mmol/L; P =2.83 10 4). exact method (14,15) as applied in the vealed the presence of the rs61736969 There was no discernible effect on fasting epitools package of the R statistical soft- C/T variation, a nonsense mutation. The plasma glucose or serum insulin concentra- ware package (R Foundation for Statistical estimated allele frequency of the T allele tions and HbA1c in homozygotes without Computing, Vienna, Austria). among the sample of 1,027 individuals diabetes when applying a recessive model The study of Nunavik Inuit was ap- was 13.2% (Supplementary Table 1). of transmission. Consistent with results proved by the Nunavik Nutrition We next assessed the allele frequencies found by Moltke et al. (3), heterozygote andHealthCommitteeandComite´ of the TBC1D4 variants among different carriers without diabetes had lower d’ethique´ de la recherche du Centre hos- ancestral populations in phase 3 of the fasting glucose and insulin concentra- pitalier de l’Universite´ de Montreal.´ The 1000 Genomes Project (8). The TBC1D4 tions (b = 20.13 mmol/L; P =2.23 24 24 GOCADAN study was approved by the c.2050C.T variant had a MAF of 0% in 10 and b = 29pmol/L;P =73 10 , ethics review board at Norton Sound Europeans, 0.6% in Latinos, 0% in African respectively). When analyzing a group Health Corporation and all relevant in- Americans, 0% in East Asians, and 0% in of 26 individuals with type 2 diabetes stitutional review boards. The analysis South Asians, and a global MAF of ;1%. It who were not using any medications investigating the risk of type 2 diabetes is important to state that rs61736969 is a for type 2 diabetes and had undergone underdiagnosis among GOCADAN partic- triallelic variant. There exists in individuals an OGTT, we found that homozygous ipants was approved by the research of African ancestry an alternate allele that carriers of the TBC1D4 variant displayed ethics committee of the Jewish General has a MAF of 3.6% and is not the same a higher 2-h plasma glucose concentra- b Hospital of Montreal. allele as the one found in Inuit. tion than noncarriers ( = +14 mmol/L; fi A detailed description of the metabolic P =0.02),buttherewasnosigni- RESULTS traits assayed for the entire GOCADAN co- cant association with 2-h serum insulin A map detailing the locations of the partici- hort, and separately for carriers, noncar- and fasting glucose and insulin con- pating Nunavik and Alaskan Inuit cohorts riers, and nongenotyped participants, centrations among these individuals appears in Fig. 1. Exome sequencing of appears in Table 1. The results of the asso- (Supplementary Table 2). – the p.Arg684Ter variant (rs61736969, ciation between the TBC1D4 p.Arg684Ter Among the rest of the type 2 diabetes a nonsense polymorphism in TBC1D4 variant and these metabolic traits for both related traits, we found a marginal de- c.2050C.T) in the cohort of 114 Nunavik the additive and recessive genetic models crease in BMI, waist circumference, and Inuit individuals (63 men and 51 women; are reported in Table 2. When comparing percentage fat mass values among hetero- mean age 52 years) revealed that the vari- homozygous carriers without diabetes of zygotes without diabetes (Table 2) and a ant was present. Of the 114 sequenced the variant with carriers of other genotypes, lower BMI among heterozygote carriers b 2 2 3 samples, 10 were removed from the we found that homozygous carriers have with diabetes ( = 5.9 kg/m ; P =1.7 1022) (Supplementary Table 2). However, we caution that these results require fur- ther replication in additional cohorts. After Bonferroni correction for multiple testing, the P value cutoff for statistical signifi- cance decreased to 3.5 3 1023.Atthis overly conservative threshold, all of our results remained significant, except for percentage fat mass and BMI. We observed consistent results when testing the association of the p.Arg684Ter variant with inverse normalized traits (Supplementary Tables 3 and 4). No association was found between the p.Arg684Ter variant and type 2 diabetes status among the 51 cases and 976 controls in the GOCADAN cohort in either the additive or recessive models (Supplementary Table 5). Figure 1—Map of the Arctic showing the regions of sampling for the Quebec Inuit (Nunavik) and Finally, we investigated how many GOCADAN (Norton Sound) cohorts. TBC1D4 carriers and noncarriers with 1892 Inuit TBC1D4 Variant and Type 2 Diabetes Diabetes Care Volume 39, November 2016

available 2-h OGTT results would have been diagnosed with prediabetes or type 2 diabetes only by meeting the 2-h OGTT diagnostic criterion. This is clinically relevant because the variant leads to in-

) Mean (SD) creased 2-h OGTT but decreased fasting n = 1,214)

n glucose, with no change in HbA1c.Among (

All participants the TBC1D4 carriers, 50 had type 2 diabetes or prediabetes, of whom 16 (32%) would have been diagnosed only by using the 2-h OGTT (30% of heterozygote carriers and 50% of homozygote carriers) (Table 3). By contrast, failure to perform a 2-h OGTT would have missed a diagnosis of type 2 diabetes or prediabetes in 16 of ) Mean (SD) Participants ( = 187) n 203 noncarriers (8%) (Table 3). The odds n ( of missing a diagnosis of type 2 diabetes or prediabetes was 5.4 (95% CI 2.5–12)

Nongenotyped participants when comparing carriers with noncarriers. Table 3 describes the rate of missed diagnoses of type 2 diabetes and prediabetes separately. Supplementary Table 6 provides the prevalences of prediabetes and type 2 diabetes in the subsample of ) Mean (SD) Participants ( = 15)

n GOCADAN participants with available n variant carriers, noncarriers, and nongenotyped participants ( OGTT results, according to their number of TBC1D4 variant copies. Homozygote carriers

TBC1D4 CONCLUSIONS Genetic disruption of TBC1D4 is present

cally in in approximately 27% of Inuit across the ﬁ North American Arctic and results in a high rate of missed diagnoses of type 2

) Mean (SD) Participants ( diabetes and/or prediabetes unless 2-h = 241) n n ( OGTTs are performed. This is because TBC1D4 disruption is associated with

Heterozygote carriers an elevation in postprandial glucose and decreases fasting glucose, while having no effect on HbA1c. This suggests that since 2-h OGTTs are rarely performed in clinical practice (16), diagnostic strategies for type 2 diabetes and prediabetes in the Inuit should now al- ) Mean (SD) Participants ( = 771) n

n ways include OGTT and/or genotypic ( Noncarriers risk stratification. These findings dem- onstrate an opportunity to apply the principles of precision medicine. Participants ( The findings confirm and extend the main findings from Moltke et al. (3) among

min) 566 115.4 (53.9) 156 110.1 (53.1) 6Greenlandic 71.9 (47.7) 88 Inuit to 104.6 (46.0) the North 816 American 113.1 (53.1)

3 Inuit. While effect sizes and directions of

mU the TBC1D4 mutation were similar in our

3 populations, the uncertain effect on

(%) type 2 diabetes risk in our population n /mmol

2 may be a result of the relatively small L number of participants with type 2 dia- 3 ) 717 27.5 (5.7) 229 26.4 (5.2) 12 26.5 (3.3) 174 27.9 (5.6) 1,132 27.3 (5.6) Characteristics of the entire GOCADAN cohort (metabolic traits) and speci 2

— betes in the GOCODAN cohort (51 cases/ (mg (%)(mmol/mol) 770 770 36.0 (5.1) 5.5 (0.5), Gutt insulin sensitivity index. 241 241976 35.0 (4.8) controls 5.4 (0.4) 15 in 15the 37.0 (3.3) GOCADAN 5.5 (0.3) 132 cohort 132 35.0 (3.6) 5.4 1,158 (0.3) 36.0 (4.9) 1,158 5.4 (0.5) 1c 1c Self-reportedDiagnosed 21 (2.7) 24 (3.1) 6 (2.5) 7 (2.9) 3 (20) 1(6.7) 3(1.6) 2 (1.1) 33 (2.7) 38 (3.1) 0,120 0,120 vs. 220 cases/1,810 controls in the Fasting plasma glucose (mmol/L)2-h plasma glucose (mmol/L)Fasting serum insulin (pmol/L)2-h serum insulin (pmol/L)HbA 771 573 768 5.3 (1.1) 570 5.2 (1.9) 65.1 (52.2) 240 205.6 (234.0) 241 156 5.1 (1.1) 156 52.2 (33.4) 5.6 (2.3) 210.0 (193.4) 15 15 6 6 5.0 (0.9) 50.2 (26.8) 350.4 (300.4) 8.9 (3.5) 133 130 91 92 5.2 (0.6) 67.2 (51.6) 227.1 (228.5) 5.6 1,159 1,154 (2.2) 823 62.5 209.9 (48.8) 5.2 (226.8) (1.0) 827 5.3 (2.0) Trait HbA ISI Table 1 Fat mass (%)Type 2 diabetes, 711 38.1 (8.2) 226 37.0 (8.3) 12 32.1 (6.2) 172 37.4 (8.8) 1,121 37.7 98.3) ISI Fasting serum triglyceride (mmol/L)Fasting serum total cholesterol (mmol/L)Fasting serum LDL cholesterol (mmol/L)Fasting serum HDL cholesterol (mmol/L) 771Albuminuria 771 (mg/g)Waist circumference 771 (cm)BMI 771 (kg/m 5.2 (1.1) 1.5 (1.1) 3.0 (0.9) 1.5 (0.5) 239 239 239 709 239 739 5.2 (1.0) 1.4 (0.6) 3.0Greenlandic (0.9) 88.0 (13.3) 1.6 (0.5) 0.1 (0.3) 15 15 227 15 15 234cohort). 5.5 (1.2) 1.7 (0.6) 84.2 (11.4) 3.1 (1.0) 0.0 1.6 (0.2) (0.3) 131 131 12 131 131 15 5.3 (1.1) 1.4 (0.9) 87.8 (6.8) 3.1 (0.9) 1.6 (0.5) 0.1 (0.3) 1,156 1,156 169 1,156 1,156 5.2 129 (1.0) 1.5 (1.0) 88.7 (13.2) 3.0 (0.9) 1.5 (0.5) 1,117 0.1 (0.2) 87.3 (13.0) 1,117 0.1 (0.2) care.diabetesjournals.org Manousaki and Associates 1893

Table 2—Association of the TBC1D4 variant with metabolic traits in individuals without diabetes from the GOCADAN cohort Additive model Recessive model Trait Patients (n) b 95% CI P value b 95% CI P value Fasting plasma glucose (mmol/L) 976 20.13 20.19 to 20.06 2.21 3 1024 20.08 0.08–0.08 0.33 2-h plasma glucose (mmol/L) 709 0.50 0.23–0.77 2.87 3 1024 3.31 0.70–4.68 2.50 3 1026

HbA1c (%) 975 20.03 20.07 to 0.01 0.10 0.13 0.08–0.28 0.10

HbA1c (mmol/mol) 975 20.3 20.8 to 0.1 0.10 1.4 0.9–3.1 0.10 Fasting serum insulin (pmol/L) 974 29.00 214.20 to 23.79 7.03 3 1024 26.84 10.83–14.39 0.53 2-h serum insulin (pmol/L) 706 13.8 220.16 to 47.85 0.43 175.28 88.65–349.03 0.05 23 ISI0,120 (SD) 703 20.15 20.31 to 0.01 0.07 21.36 22.18 to 20.53 1.21 3 10 Fasting serum HDL cholesterol (mmol/L) 974 0.02 20.05 to 0.08 0.64 0.09 0.13–0.35 0.46 Fasting serum LDL cholesterol (mmol/L) 974 0.01 20.12 to 0.13 0.93 0.09 0.26–0.60 0.73 Fasting serum triglyceride (mmol/L) 974 20.08 20.20 to 0.04 0.18 0.32 0.25–0.80 0.20 Fasting serum total cholesterol (mmol/L) 974 0.00 20.13 to 0.14 0.98 0.35 0.28–0.89 0.21 Waist circumference (cm) 919 22.85 24.70 to 20.99 2.47 3 1023 20.10 4.11–7.98 0.98 Fat mass (%) 921 20.99 21.73 to 20.25 0.01 20.61 1.64–2.60 0.71 BMI (kg/m2)93120.83 21.63 to 20.04 0.04 20.86 1.79–2.64 0.63 Albuminuria (mg/g) 939 0.27 20.06 to 0.60 0.11 3.38 3.31–9.86 0.31 Results are shown for an additive and a recessive genetic model. The number of patients for each trait includes those with genotype data for the specific variant and phenotypic data for the specific trait. b is the effect size (change in the trait expressed in its unit of measurement if one copy of the effect allele is present) estimated using untransformed values. All P values ,0.05 are considered statistically significant and appear in boldface. ISI0,120, Gutt insulin sensitivity index.

There is a plausible pathophysiologic postprandial glucose seems to be a bet- diagnostic criteria for type 2 diabetes explanation for the abovementioned ter predictor of cardiovascular disease (13,24). However, all the above-mentioned findings. TBC1D4 acts as a mediator of and all-cause mortality than fasting studies have been done in non-Inuit pop- insulin-stimulated glucose uptake through glucose, and this association is linear ulations, leaving open the possibility that GLUT4 mobilization (17). Murine ex- (20,21). Specifically, postprandial glu- the impact of postprandial hyperglyce- periments have shown that knocking cose .7.8 mmol/L has been associated mia might be different in Inuit carrying down Tbc1d4 causes a significant de- with all-cause mortality (22), and post- the TBC1D4 variant. crease in basal plasma glucose and insulin- prandial glucose .10.0 mmol/L has Our findings suggest that the TBC1D4 stimulated glucose uptake in muscle been associated with microvascular variant is relatively common among the and adipose tissue (18) In humans, complications and myocardial infarc- Eastern Canadian Nunavik Inuit and the these findings are supported by a case tions (23). Some of these studies found Alaskan Inuit and potentially in other Arctic report of familial postprandial hyper- that these effects of postprandial glu- populations. These findings are clinically insulinemia associated with a TBC1D4 coseoccurredinadditiontoeffects relevant given the increasing prevalence variant (19). from fasting glucose and HbA1c.These of diabetes in these populations (6,25,26). Carryingamutationthat solely increases findings and others have prompted the With an expected allele frequency of ap- postprandial glucose may influence type 2 American and Canadian Diabetes Asso- proximately 14%, we anticipate that 2% of diabetes complications. Interestingly, ciations to include OGTT as part of the the North American Inuit population are

Table 3—Classiﬁcation of TBC1D4 variant carriers and noncarriers according to their type 2 diabetes and prediabetes status and their diagnostic criteria for type 2 diabetes T2D or prediabetes T2D Prediabetes Only 2-h FG T2D and prediabetes Only 2-h FG Only 2-h FG glucose and/or cases diagnosed only glucose and/or glucose and/or

Criteria for T2D diagnosis (OGTT) HbA1c by OGTT (%) (OGTT) HbA1c (OGTT) HbA1c TBC1D4 noncarriers 16 187 8 7 11 9 176 TBC1D4 heterozygote carriers 14 32 30 4 3 10 29 TBC1D4 homozygote carriers 2 2 50 1 0 1 2 TBC1D4 carriers (homozygote + heterozygote) 16 34 32 5 3 11 31 Data are n unless otherwise indicated. The numbers of individuals with only abnormal 2-h plasma glucose on the OGTT (according to the American Diabetes Association criteria for type 2 diabetes [T2D] and prediabetes) represent cases of missed T2D or prediabetes diagnoses. FG, fasting glucose. 1894 Inuit TBC1D4 Variant and Type 2 Diabetes Diabetes Care Volume 39, November 2016

homozygous and 25% are heterozygous for heterozygous carriers could then be fur- References the TBC1D4 variant. Among the possible ther investigated by OGTTs. Of note, ge- 1. Hudson KLR, Patrick-Lake B, Burchard EG, public health implications of this finding, netic screening tests for colon cancer have et al.; Precision Medicine Initiative (PMI) Work- we speculate that ascertainment of the been successfully implemented in the ing Group. The Precision Medicine Initiative Cohort Program - Building a Research Founda- TBC1D4 variant could serve as a useful Quebec Inuit population (28). tion for 21st Century Medicine. Bethesda, MD, screening test to predict patients with An important limitation of this study National Institutes of Health, 2015 prediabetes and type 2 diabetes with was that the degree of admixture of the 2. Collins FS, Varmus H. A new initiative on pre- pronounced insulin resistance and exclu- Inuit population studied could not be cision medicine. N Engl J Med 2015;372:793–795 sively abnormal postprandial glucose quantified. Moltke et al. (3) showed an 3. Moltke I, Grarup N, Jørgensen ME, et al. A fl common Greenlandic TBC1D4 variant confers concentrations. important in uence of admixture on the muscle insulin resistance and type 2 diabetes. Theclinicalrelevanceofthesefindings is MAF of the TBC1D4 variant among Green- Nature 2014;512:190–193 magnified since 2-h OGTTs are rarely per- landic Inuit, but the degree of admixture 4. Jørgensen ME, Bjeregaard P, Borch-Johnsen formed in clinical practice; type 2 diabetes among the GOCADAN population cannot K. Diabetes and impaired glucose tolerance among the inuit population of Greenland. Dia- and prediabetes are generally diagnosed be likewise assessed because of a lack of betes Care 2002;25:1766–1771 using fasting glucose, random glucose, and dense genotyping. Nonetheless, this does 5. Helgason A, Palsson´ G, Pedersen HS, et al. HbA1c (13,24,27). Although the frequency not change the clinical relevance of these mtDNA variation in Inuit populations of Green- of OGTT among the Inuit population has findings, since an important proportion of land and Canada: migration history and popula- not been reported, a population-based reg- the North American Inuit population car- tion structure. Am J Phys Anthropol 2006;130: 123–134 istry of laboratory tests for type 2 diabetes ries the TBC1D4 variant. 6. Howard BV, Devereux RB, Cole SA, et al. A diagnosis in Ontario, Canada (16), showed Detecting the presence of the TBC1D4 genetic and epidemiologic study of cardiovascu- that over 20 years (1995–2005) there variant in the Inuit population might be lar disease in Alaska natives (GOCADAN): design was a 28% increase in fasting glucose important not only for diagnostic pur- and methods. Int J Circumpolar Health 2005;64: – tests, which were prescribed for 37% of poses but also for therapeutic decision 206 221 7. Zhou S, Xiong L, Xie P, et al. Increased mis- adults without known type 2 diabetes in making. TBC1D4 carriers represent a sub- sense mutation burden of fatty acid metabolism 2005. During the same period, the per- group of patients with type 2 diabetes related genes in Nunavik Inuit population. PLoS centage of HbA1c tests also increased who may be good candidates for specific One 2015;10:e0128255 from 1.7% in 1995 to 6.0%. Remarkably, pharmacologic treatments. This is because 8. Abecasis GR, Altshuler D, Auton A, et al.; less than 1% of this population under- the mechanism of action of TBC1D4 could 1000 Genomes Project Consortium. A map of human genome variation from population-scale went OGTT testing in any year between suggest a better response of carriers to sequencing [published correction appears in 1995 and 2005, although OGTT has been insulin sensitizers (29,30) than to other Nature 2011;473:544]. Nature 2010;467:1061– recommended for type 2 diabetes screen- first-line type 2 diabetes oral antihypergly- 1073 ing by the Canadian Diabetes Association cemic drugs, such as sulfonylureas. How- 9. Gutt M, Davis CL, Spitzer SB, et al. Validation since 2003 (24). Thus current clinical prac- ever, this hypothesis should be tested in of the insulin sensitivity index (ISI(0,120)): com- fi parison with other measures. Diabetes Res Clin tice would miss an important proportion clinical trials. Finally, our ndings could be Pract 2000;47:177–184 of the Inuit population with type 2 diabe- applied in pharmacologic research, as 10. Blangero J, Goring HH, Kent JW Jr, et al. tes unless OGTTs are performed. TBC1D4 may represent a potential drug Quantitative trait nucleotide analysis using We did not observe a significant in- target for type 2 diabetes. Bayesian model selection. Hum Biol 2005;77: – crease of missed type 2 diabetes diagno- In summary, genetic disruption of 541 559 11. Almasy L, Blangero J. Multipoint quantitative- ses without OGTT among TBC1D4 carriers, TBC1D4 is common across the North trait linkage analysis in general pedigrees. Am J likely because of the small sample of pa- American Inuit and leads to a higher Hum Genet 1998;62:1198–1211 tients with type 2 diabetes and OGTT val- postprandial glucose. Type 2 diabetes 12. Gelman A, Hill J, Yajima M. Why we (usually) ues (n = 26) in the GOCADAN cohort; and prediabetes should be assessed in don’t have to worry about multiple compari- – nevertheless, we found a sevenfold in- the population using OGTT, either after sons. J Res Educ Eff 2012;5:189 211 fi 13. American Diabetes Association. Diagnosis crease of the odds of missing a diagnosis genotypic risk strati cation or by test- and classification of diabetes mellitus. Diabetes of prediabetes. Considering that TBC1D4 ing the entire at-risk population. Care 2010;33(Suppl. 1):S62–S69 carriers with prediabetes and diabetes 14. Mehta CR, Patel NR. A hybrid algorithm for (27% of the Inuit population) have a five- Fishers exact test in unordered rxc contingency- fl fold increase in their combined risk to re- Duality of Interest. No potential con icts of tables. Commun Stat Theory Methods 1986;15: interest relevant to this article were reported. 387–403 main undiagnosed (or a 32% chance to Author Contributions. D.M., J.W.K., K.H., D.E.N., 15. Clarkson DB, Fan YA, Joe H. A remark on remain undiagnosed) without measuring J.G.U., A.G.C., and J.B.R. conducted the analyses. algorithm-643 - Fexact - an algorithm for per- 2-h glucose, performing OGTTs for these D.M. and J.B.R. wrote the first draft of the man- forming Fishers exact test in R X C contingency- Inuit individuals seems to be of clinical uscript. J.W.K., K.H., D.E.N., J.G.U., and A.G.C. tables. ACM Trans Math Software 1993;19: – relevance. Given that performing an conducted the sequencing of the TBC1D4 variant 484 488 in the GOCADAN cohort. S.Z., P.X., and G.R. con- 16. Wilson SE, Lipscombe LL, Rosella LC, OGTT as a screening test for type 2 di- ducted the sequencing of the TBC1D4 variant in Manuel DG. Trends in laboratory testing for diabetes and prediabetes can beparticularly the Quebec Inuit cohort. J.B.R. conceived the abetes in Ontario, Canada 1995–2005: a popu- cumbersome in the Inuit population, ge- experiment. C.M.G., P.B., and S.C. provided im- lation-based study. BMC Health Serv Res 2009; netic screening for the TBC1D4 mutation portant feedback on the manuscript. J.B.R. is 9:41 the guarantor of this work and, as such, had 17. Sano H, Kane S, Sano E, et al. Insulin-stimulated might represent a clinically useful alterna- full access to all the data in the study and takes phosphorylation of a Rab GTPase-activating protein tive, especially if the cost-effectiveness of responsibility for the integrity of the data and regulates GLUT4 translocation. J Biol Chem 2003; this approach is shown. 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