Type 2 diabetes susceptibility gene variants predispose to adult-onset autoimmune diabetes
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Latent autoimmune diabetes in adults (LADA) is phenotypically a hybrid of type 1 and type 2 diabetes. Genetically LADA is poorly characterised but does share genetic predisposition with type 1 diabetes. We aimed to improve the genetic characterisation of LADA and hypothesised that type 2 diabetes-associated gene variants also predispose to LADA, and that the associations would be strongest in LADA patients with low levels of GAD autoantibodies (GADA).
We assessed 41 type 2 diabetes-associated gene variants in Finnish (phase I) and Swedish (phase II) patients with LADA (n = 911) or type 1 diabetes (n = 406), all diagnosed after the age of 35 years, as well as in non-diabetic control individuals 40 years or older (n = 4,002).
Variants in the ZMIZ1 (rs12571751, p = 4.1 × 10−5) and TCF7L2 (rs7903146, p = 5.8 × 10−4) loci were strongly associated with LADA. Variants in the KCNQ1 (rs2237895, p = 0.0012), HHEX (rs1111875, p = 0.0024 in Finns) and MTNR1B (rs10830963, p = 0.0039) loci showed the strongest association in patients with low GADA, supporting the hypothesis that the disease in these patients is more like type 2 diabetes. In contrast, variants in the KLHDC5 (rs10842994, p = 9.5 × 10−4 in Finns), TP53INP1 (rs896854, p = 0.005), CDKAL1 (rs7756992, p = 7.0 × 10−4; rs7754840, p = 8.8 × 10−4) and PROX1 (rs340874, p = 0.003) loci showed the strongest association in patients with high GADA. For type 1 diabetes, a strong association was seen for MTNR1B (rs10830963, p = 3.2 × 10−6) and HNF1A (rs2650000, p = 0.0012).
LADA and adult-onset type 1 diabetes share genetic risk variants with type 2 diabetes, supporting the idea of a hybrid form of diabetes and distinguishing them from patients with classical young-onset type 1 diabetes.
KeywordsAdult-onset type 1 diabetes GADA GAD autoantibodies Genetics HNF1A LADA Latent autoimmune diabetes in adults MTNR1B TCF7L2 ZMIZ1
All New Diabetics in Scania
Diabetes Registry in Vasa
Latent autoimmune diabetes in adults
Malmö Diet and Cancer Study
Diabetes is a complex disease with two main subtypes: type 1 diabetes, characterised by autoimmune destruction of the insulin-producing beta cells, and type 2 diabetes, characterised by a combination of insulin resistance and reduced insulin secretion. The subdivision into type 1 and type 2 diabetes is especially intricate in adults, in whom the disease is more like a spectrum of phenotypes. This spectrum also includes the subtype termed latent autoimmune diabetes in adults (LADA).
LADA accounts for about 10% of European patients initially diagnosed with type 2 diabetes in whom type 1 diabetes-associated autoantibodies are present [1, 2, 3]. The most prevalent autoantibodies among these adults are GAD autoantibodies (GADA), present in 90%. Other autoantibodies, such as those to protein tyrosine phosphatase IA-2 or zinc transporter 8 are reported in only 1–2% of GADA-negative patients [3, 4]. The level of autoantibodies, particularly GADA, has a clear influence on the phenotype of LADA. A high GADA level is associated with a phenotype more like type 1 diabetes, including reduced beta cell function, increased need for insulin treatment, lower BMI and lower prevalence of dyslipidaemia (reviewed by [5, 6]).
The genetic component of diabetes has been intensively studied in patients with type 2 diabetes and in patients with type 1 diabetes diagnosed in childhood, which has resulted in identification of several susceptibility gene variants . Genetic studies in LADA comprising >100 patients are rare but indicate that LADA shares a genetic predisposition with childhood-onset type 1 diabetes. Association with HLA-DQB1 and PTPN22 risk alleles has consistently been shown, while association with INS is disputed. Furthermore, the frequency of these type 1 diabetes risk alleles has consistently been shown to correlate with GADA level among LADA patients (reviewed by ). For type 1 diabetes the genetic predisposition seems to be similar in children and adults diagnosed predominantly at >15 years of age; only for HLA-DQB1 and IL2RA have effects related to age at onset been reported [8, 9, 10].
Whether type 2 diabetes-associated variants predispose to adult-onset type 1 diabetes or LADA is largely unknown. To date, consistent association has only been reported for TCF7L2 and LADA [11, 12]. We hypothesised that LADA, and possibly adult-onset type 1 diabetes, is a genetic hybrid of type 1 and type 2 diabetes with increased frequency of type 2 diabetes risk genotypes. Further, we expected the type 2 diabetes-associated gene variants to show strongest association in LADA patients with low GADA levels, whose disease is phenotypically more like type 2 diabetes. In this study, we assessed the association of type 2 diabetes susceptibility gene variants in LADA and adult-onset type 1 diabetes diagnosed at age >35 years, to explore the degree of genetic overlap in adult-onset forms of diabetes.
We included patients with LADA or type 1 diabetes diagnosed at >35 years of age and non-diabetic control individuals. The control individuals were aged ≥ 40 years at the time of investigation (Finnish/Swedish, mean ± SD, 56.6 ± 10.3/56.8 ± 6.2 years), had a BMI of 26.5 ± 4.0/25.4 ± 3.7 kg/m2 and included 46.1/48.8% men.
Diagnosis of LADA was based on presence of GADA and sufficient beta cell function at the time of diagnosis, indicated by no insulin treatment and/or C-peptide level >0.2 nmol/l. The majority of the LADA patients had initially been diagnosed with type 2 diabetes. Diagnosis of type 1 diabetes was based on initial diagnosis of type 1 diabetes made by the treating physician, a fasting serum C-peptide concentration< 0.2 nmol/l at the time of investigation and initiation of permanent insulin treatment within 6 months from diagnosis.
Measurements and assays
C-peptide concentrations were determined by a radioimmunoassay (Human C-peptide RIA; Linco, St Charles, MO, USA; or Peninsula Laboratories, Belmont, CA, USA), or with Cobas e411 (Roche Diagnostics, Mannheim, Germany). GADA were measured with an ELISA assay (RSR, Cardiff, UK) in the DIREVA patients (positive cut-off: 10 IU/ml) and with radiobinding assays using 35S-labelled protein  in the rest of the patients (positive cut-off: 5 RU or 32 IU/ml). The radiobinding assay showed 62–88% sensitivity and 91–99% specificity in workshops (Combinatorial Autoantibody or Diabetes/Islet Autoantibody Standardization Programs) from 1998 to 2013, and the ELISA assay showed 72% sensitivity and 99% specificity in the 2013 Islet Autoantibody Standardization Program workshop.
To assess the relationship between gene variants and GADA level, the LADA patients were stratified into LADAhigh and LADAlow according to the median GADA level among Finnish LADA patients. The same cut-off values (Botnia, 20.4 RU or 73.0 IU/ml; DIREVA, 59.0 IU/ml) were applied to the Swedish patients (65.9% were LADAhigh) as they were typed using the same methods.
All cases (n = 1,317) and a subset of the controls (n = 987) were genotyped with a single-nucleotide polymorphism (SNP) panel, custom-designed for the All New Diabetics in Scania (ANDIS) study in Sweden (http://snd.gu.se/en/catalogue/study/EXT0057; accessed 7 April 2014), including SNPs reported to be associated with type 2 diabetes or related phenotypes. Genotyping was performed by matrix-assisted laser desorption–ionisation time-of-flight mass spectrometry on a MassARRAY platform (Sequenom, San Diego, CA, USA). We excluded SNPs with a minor allele frequency (MAF) < 5%, with genotyping success rate < 90% or disagreeing with Hardy–Weinberg equilibrium (p < 0.00029, corresponding to 0.05/No. of SNPs). Individuals with missing genotypes for >5% of the SNPs were also excluded. Altogether, 143 SNPs (genotyping success rate > 99%), 1,272 cases and 905 control individuals passed the quality control.
The rest of the controls (Botnia, n = 873; MDC, n = 2,142) had been genotyped with the Metabochip , where genotypes were called with GenomeStudio (Illumina, San Diego, CA, USA). SNPs included both on the Metabochip and in the ANDIS panel numbered 105; of these SNPs, 46 were in or near loci previously associated with type 2 diabetes in Europeans  and 40 passed the quality control (electronic supplementary material [ESM] Table 1). In addition, we included a SNP in GLIS3, as this is one of the few loci that have shown an association with both type 1 and type 2 diabetes (type 2 diabetes association only in Asians). Hence, 41 SNPs were analysed.
Statistical Package for Social Science software (version 19.0; SPSS, Chicago, IL, USA) was applied to compare clinical characteristics between patient groups by the non-parametric Mann–Whitney U test or the χ2 test. Genetic association analyses were performed with PLINK v1.07 (http://pngu.mgh.harvard.edu/~purcell/plink/ ). Differences in allele frequencies between cases and controls were assessed with logistical regression adjusted for the effect of age, sex and BMI. ORs are presented for the reported type 2 diabetes-associated alleles. For the association tests in the Finnish phase I data set a p value of < 0.0012 was considered statistically significant, corresponding to a Bonferroni correction for the number of SNPs tested (n = 41). RevMan v5.2.6 (http://tech.cochrane.org/revman/download) was applied to conduct fixed-effect meta-analyses estimating the combined effect sizes for the Finnish and Swedish data sets. Cochran’s Q test was used to assess the heterogeneity between data sets in the meta-analyses.
The possible effect of HLA-DQB1 risk genotypes (*0302 or *0302/02) on association results was tested in part of the Finnish data set with available HLA-DQB1 genotype data (LADA, n = 264; type 1 diabetes, n = 245; control individuals, n = 503). HLA-DQB1 genotype was included as covariate in logistical regression analyses; the main results were similar with this adjustment (data not shown).
Type 1 diabetes
Men, n (%)
Age at diagnosis, years
Age at investigation, years
fS C-peptide, nmol/l
Total cholesterol, mmol/l
Phase I: Finnish patients
Association results: LADA
SNP – Candidate gene
OR (95% CI)
rs12571751 – ZMIZ1
1.35 (1.15, 1.60)
3.2 × 10−4a
rs7903146 – TCF7L2
1.44 (1.18, 1.77)
3.8 × 10−4a
rs10842994 – KLHDC5
1.44 (1.15, 1.80)
rs896854 – TP53INP1
1.28 (1.09, 1.50)
rs108116661 – CDKN2A/B
1.42 (1.11, 1.80)
rs10830963 – MTNR1B
1.27 (1.07, 1.51)
rs1111875 – HHEX
1.23 (1.05, 1.46)
rs2237895 – KCNQ1
1.20 (1.02, 1.41)
rs7578597 – THADA
1.49 (1.03, 2.16)
rs7756992 – CDKAL1
1.20 (1.01, 1.43)
rs7754840 – CDKAL1
1.19 (1.01, 1.40)
Adult-onset type 1 diabetes
Association results: adult-onset type 1 diabetes
SNP – Candidate gene
OR (95% CI)
rs10830963 – MTNR1B
1.59 (1.32, 1.93)
1.7 × 10−6a
rs340874 – PROX1
0.68 (0.56, 0.82)
6.1 × 10−5a
rs2650000 – HNF1A
1.43 (1.19, 1.72)
1.7 × 10−4a
rs7754840 – CDKAL1
1.26 (1.05, 1.52)
rs7756992 – CDKAL1
1.26 (1.04, 1.53)
rs10811661 – CDKN2A/B
1.38 (1.05, 1.80)
rs864745 – JAZF1
0.81 (0.67, 0.97)
rs10401969 – CILP2
0.64 (0.43, 0.96)
rs7957197 – HNF1A
1.27 (1.02, 1.57)
Phase II: Swedish patients and meta-analyses
SNPs obtaining a p value <0.05 (adjusted for age, sex and BMI) in phase I were tested in the Swedish phase II data set.
The ZMIZ1 association was replicated in the Swedish LADA patients (1.27 [1.08, 1.50], p = 0.0049), but driven by LADAhigh (1.40 [1.15, 1.70], p = 7.2 × 10−4; LADAlow: 1.02 [0.76, 1.37], p = NS). The TCF7L2 risk allele was significantly increased in the Swedish LADAlow patients (1.55 [1.13, 2.13], p = 0.0066), but failed to reach statistical significance in the whole group. None of the other associations observed in Finnish LADA patients were replicated in the Swedish patients (ESM Table 1).
In meta-analyses where LADA patients were stratified based on GADA level, association with LADAlow was accentuated for TCF7L2 (p = 2.4 × 10−4) and KCNQ1 (p = 0.0012) (Fig. 3) and suggestive association was observed for MTNR1B (1.27 [1.08, 1.50], p = 0.0039). With respect to LADAhigh, ZMIZ1 (p = 1.1 × 10−4) and CDKAL1 (rs7756992, p = 7.0 × 10−4; rs7754840, p = 8.8 × 10−4) showed significant association below the Bonferroni-corrected threshold (Fig. 3) and suggestive association was seen for PROX1 (rs340874, 1.22 [1.07, 1.39], p = 0.003) and TP53INP1 (rs896854, 1.21 [1.06, 1.37], p = 0.005). Only the TP53INP1 SNP showed signs of heterogeneity between the data sets (p = 0.09).
Adult-onset type 1 diabetes
To improve the genetic characterisation of LADA and to address the question of genetic overlap in the adult-onset forms of diabetes, we studied type 2 diabetes-associated gene variants in patients with LADA or adult-onset type 1 diabetes diagnosed at > 35 years of age. We show that both subgroups of adult-onset autoimmune diabetes share genetic risk variants with type 2 diabetes. The reported associations show effect sizes and direction of effect consistent with those reported for type 2 diabetes, which indicate that the associations are true. With respect to LADA, the level of GADA had a distinct effect on the genetic associations. Previously, we have assessed type 1 diabetes-associated gene variants in the LADA patients included in the current study and reported association with HLA-DQB1 and PTPN22, although with lower risk-allele frequencies in the LADA patients than in patients with young and adult-onset type 1 diabetes [9, 11]. LADA, thus, seems to be a genetic hybrid of type 1 and type 2 diabetes distinct from classical young-onset type 1 diabetes.
A great strength of our study is the inclusion of a unique collection of patients with adult-onset type 1 diabetes diagnosed after the age of 35 years, and the largest published collection of genetically characterised LADA patients, numbering over 900. Further, we analysed LADA and adult-onset type 1 diabetes as separate phenotypes since type 1 diabetes risk variants show different frequencies in the two diabetes subtypes. Even though this stratification reduces the sample size in the groups analysed, the more precise phenotyping may pinpoint associations that would be missed in the combined group of unspecified adult-onset autoimmune diabetes.
The sample size is a limitation of our study, especially when considering the reported effect sizes of variants associated with type 2 diabetes ranging from OR of 1.05 to 1.38 . Thus, we can detect the variants showing strongest association with LADA or adult-onset type 1 diabetes but lack statistical power to exclude association with other variants. This problem was accentuated in the analyses of GADA-level subgroups. Another limitation is the fact that our study is based on current knowledge of the genetic predisposition to type 2 diabetes. For the great majority of associated loci the causal variant is still unknown and therefore most of the examined variants represent statistical association and not necessarily biological association. The lack of knowledge about the causal variant within each locus is reflected in the difficulties of pinpointing the mechanistic links between genetic variants and disease.
Variants in TCF7L2 and ZMIZ1 showed strong evidence of association with LADA. In Europeans, the intronic TCF7L2 rs7903146 variant is the strongest identified genetic risk factor for type 2 diabetes . Association has also been shown between TCF7L2 and LADA  in work encompassing a subset of the patients included in the current study . TCF7L2 encodes a transcription factor, which is involved in maintaining the secretory function of beta cells and has been suggested to be a key determinant of beta cell mass (reviewed in ). The rs7903146 variant is associated with impaired insulin secretion, although the exact mechanistic link is unclear . The variant most likely imposes a non-autoimmune reduction in insulin secretion, which in LADA patients exacerbates the presumably already reduced beta cell function caused by autoimmune destruction.
In agreement with the Italian Non-Insulin Requiring Autoimmune Diabetes (NIRAD) study , the meta-analysis showed stronger TCF7L2 association in patients with low GADA level. This GADA-level effect was, however, driven by the Swedish data set, while the association in the Finnish data set was independent of GADA level. This discrepancy may reflect differences in the differentiation between LADA and type 1 diabetes in Finnish and Swedish patients. The frequency of the TCF7L2 risk T allele among the adult-onset type 1 diabetes patients and control individuals was similar, in line with previous reports in a mixture of patients with adult-onset autoimmune diabetes  and in childhood-onset type 1 diabetes . Thus, TCF7L2 represents a distinguishing factor between LADA and type 1 diabetes, irrespective of age at diagnosis, and the T allele seems to protect against type 1 diabetes possibly by sufficient impairment of insulin synthesis, resulting in reduced antigenic burden suggested to be operative in type 1 diabetes.
Importantly, the association of ZMIZ1 rs12571751 with LADA is novel. An association was seen both in Finnish and Swedish LADA patients, but in the Swedish patients it was strongest in those with a high GADA level. Other intronic ZMIZ1 variants have been associated with a variety of autoimmune diseases, including childhood-onset type 1 diabetes  and coeliac disease . Even though we observed no association between the variant and adult-onset type 1 diabetes, the ZMIZ1 loci seems to play a key role in the development of autoimmune diseases.
ZMIZ1 is an interesting candidate gene for LADA as the encoded protein is a transcriptional co-activator interacting with Smad3 and p53 [29, 30]. Both these transcription factors are involved in pathways possibly linked to diabetes susceptibility. Smad3 modulates the TGF-β signalling pathway, which is linked to immune tolerance, inhibition of autoimmune diabetes and induction of anti-inflammatory responses [31, 32]. On the other hand, p53 is linked to apoptosis, which may be a determining factor for beta cell mass but also in the autoimmune process where apoptosis may induce the formation of autoantibodies by increasing the exposure to autoantigens . The p53 link is also interesting in light of the suggestive association between TP53INP1 and LADAhigh, as TP53INP1 is a direct target gene for p53 and has been suggested to regulate p53-dependent apoptosis . Moreover, reduced activity of transcription factor 7-like 2 has been linked to increased expression of TP53INP1 and beta cell apoptosis . Thus, p53 and apoptosis could provide a molecular connection between three LADA-associated genes.
We hypothesised that among LADA patients, those with low GADA levels would have a genotype more similar to type 2 diabetes (i.e. they show higher frequencies of the type 2 diabetes risk variants than those with high GADA levels). However, only a few type 2 diabetes variants supported this hypothesis and the results were inconsistent in the Finnish and Swedish data sets. This heterogeneity in GADA-level association may, of course, be explained by the small sample size. An alternative explanation is that the associations, which seem to be GADA-level dependent, are mediated by factors that correlate with the GADA level, such as BMI, and that the type 2 diabetes variants are not directly linked to the autoimmune process in LADA. Considering LADA, it is, however, very interesting that some of the type 2 diabetes variants are associated only with LADAhigh, as this supports LADA being a true hybrid of type 1 diabetes and type 2 diabetes and not just a mixed group of misclassified patients with type 1 diabetes or type 2 diabetes.
With respect to type 1 diabetes, we hypothesised that non-autoimmune mechanisms may contribute to disease development, especially in adult-onset type 1 diabetes where the autoimmune disease process seems less pronounced. A genetic overlap between type 1 and type 2 diabetes is supported by the reported associations for variants in ZMIZ1 and GLIS3 with both subtypes of diabetes [20, 27, 35] and possibly also by associations for variants in HHEX and PPARG, for which inconsistent results have been published [36, 37]. Our data showed no support for these previous findings, as none of the four loci were associated with adult-onset type 1 diabetes and only ZMIZ1 was associated with LADA. Of note, with the current sample size we were unable to rule out association for these loci. However, in support of the hypothesis of a genetic overlap between type 1 and type 2 diabetes, we identified novel associations between adult-onset type 1 diabetes and variants in two type 2 diabetes-associated loci, MTNR1B and HNF1A. Both associations were driven by the Finnish patients, but remained significant in the meta-analysis. The discrepancy between the data sets may be due to the small sample size or the significant difference between Finnish and Swedish patients with respect to age at diagnosis.
The intronic MTNR1B rs10830963 variant has been associated with type 2 diabetes, elevated fasting glucose level and impaired insulin response to oral and intravenous glucose. MTNR1B encodes the melatonin receptor 1B (MT2), which is expressed in human pancreatic islets and beta cells and plays a key role in the regulation of circadian rhythms. Disruption of the circadian rhythms has been linked to metabolic disorders such as type 2 diabetes (reviewed by ). Furthermore, melatonin has been shown to be an immune stimulator (reviewed by ) and seems to be directly involved in the modulation of insulin secretion. However the effect of melatonin on insulin secretion is debatable, as melatonin has been shown to inhibit insulin secretion in vivo and in vitro in rodent models, whereas in one study it enhanced insulin secretion in human islets [40, 41].
The functional link between MTNR1B and type 2 diabetes is unclear. The mechanism has been suggested to be gain-of-function, as the rs10830963 risk allele is associated with increased mRNA expression of the MT2 receptor in islets from non-diabetic individuals . In contrast, studies of rare coding MTNR1B variants point to loss of receptor function as the causal mechanism linked to type 2 diabetes .
A role for MTNR1B in the pathogenesis of autoimmune diabetes is further supported by a possible association between the MTNR1B variant and LADA, especially in the Finnish patients, in whom the risk-allele frequency was similar in LADAhigh and LADAlow patients. However, as no association has been observed between the variant and childhood-onset type 1 diabetes , these data may indicate that MTNR1B is a candidate gene only for the adult-onset forms of diabetes. This hypothesis is supported by the observed age-related reduction in endogenous melatonin levels. Reduced melatonin levels have been linked to immunosenescence, a process that is thought to contribute to autoimmune diseases in elderly people (reviewed by ).
Rare mutations in HNF1A cause MODY , and the common variants have been linked to LDL-cholesterol (rs2650000) and type 2 diabetes (rs7957197) [45, 46]. HNF1A encodes a transcription factor essential for pancreatic beta cell development and function  and is thus a good candidate gene for diabetes in general. This HNF1A–type 1 diabetes association, together with the majority of the LADA associations, point to compromised beta cell mass as a key mechanism contributing to insulin deficiency also in adult-onset autoimmune diabetes.
Our study adds to the genetic knowledge about the much-debated diabetes subtype LADA. However, so far all genetic studies of LADA have been candidate-gene based and have focused on type 1 or type 2 diabetes-associated variants, thus it is still unknown whether LADA harbours its own unique risk variants. A genome-wide association study in a larger sample of LADA patients will be needed to answer this question. A larger sample size would also permit extended stratification of the LADA patients according to HLA genotype, which could reveal interesting genetic interactions, and according to the presence of additional autoantibodies, which could reveal genetic differences as carriers of multiple autoantibodies show a more rapid disease progression.
The Botnia Research Group, the FinnDiane Research group, the Västerbotten Study group and the DIREVA study group are acknowledged for recruiting and clinically studying the participants.
The Botnia Study was supported by grants from the Academy of Finland (263401 and 267882), the Sigrid Juselius Foundation, the Finnish Diabetes Research Foundation, the Folkhälsan Research Foundation, the Finska Läkaresällskapet, the Ollqvist Foundation, Korsholm, Malax, Närpes, and Vasa Health Care Centers and The Helsinki University Central Hospital. The FinnDiane Study was supported by grants from the Folkhälsan Research Foundation, the Wilhelm and Else Stockmann Foundation and the Academy of Finland. The Västerbotten Study was funded by the Västerbotten County Council. The MDC study was supported by a project grant from the Swedish Research Council (K2011-65X-20752-04-6). The ANDIS Study was supported by infrastructure grants from the Swedish Research Council (2010-5983 and 2012-5538). LG was supported by an ERC Advanced Research Grant GENETARGET-T2D (GA 269045).
Duality of interest
P-HG has received research grants from Eli Lilly and Roche, as well as lecture honoraria from Boehringer Ingelheim, Eli Lilly, Genzyme, Medscape, MSD, Novartis and Novo Nordisk. P-HG is an advisory board member of Abbott, AbbVie, Boehringer Ingelheim, Cebix, Eli Lilly and Novartis. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.
The study was planned by MKA, OR, P-HG, LG and TT. MS, TF, AK, CF, KL and PMN contributed to phenotype and genotype data acquisition. MKA and TT analysed and interpreted the data and drafted the initial version of the manuscript. All co-authors reviewed the manuscript and approved the submitted version. MKA is the guarantor of this work.
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