A Loss-of-Function Splice Acceptor Variant in IGF2 Is Protective for Type 2 Diabetes

Abstract

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage-dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

Figure 1

Discovery and replication of the rs149483638 T2D protective variant. Quantile-quantile plot for all common variants (A) and for Mexican population–specific variants (B). The plot shows the two most significant variants that have low frequency in Europeans but higher frequency in the Mexican population. C: Forest plot for the meta-analysis of rs149483638 variant in IGF2. We replicated the rs149483638 association in four independent data sets: 1,007 T2D case and 917 control subjects of Hispanic origin from T2D-GENES (MAF = 0.12, OR = 0.89, P = 0.3), 1,519 T2D case and 1,680 control subjects of full-heritage American Indian ancestry from the Pima cohort (MAF = 0.14, OR = 0.68, P = 1.09 × 10⁻⁵), 427 case and 751 control subjects of self-identified indigenous individuals from different ethnic groups in Mexico (DMS2 cohort) (MAF = 0.36, OR = 0.71, P = 0.001), and 1,064 case and 4,832 control subjects from the subset of subjects with Latino ancestry from the GERA cohort (MAF = 0.06, OR = 0.82, P = 0.11).

Figure 2

rs149483638 prevents splicing in vitro. A: This variant is located at a canonical splice acceptor site and is predicted to cause skipping of exon 2 of IGF2 isoform 2. B: 293T cells were transfected with IGF2 minigenes containing the first three exons and two introns of the IGF2 gene, and either allele of the rs149483638 C>T variant (G>A in the reverse strand) and cDNA were analyzed by ddPCR. This analysis revealed no expression of the IGF2 exon 1-2 junction in cells transfected with the minigene containing the T2D-protective rs149483638 A allele. This was in contrast to the high levels of exon 1-2 splicing detected in cells transfected with the G allele. C: One-dimensional plots of the ddPCR droplets plotted in B. No IGF2 transcript was detected in untransfected samples. ACTB was used as an internal control.

Figure 3

rs149483638 prevents splicing between exon 2 in liver and in adipose tissue. The dosage of the T2D-protective A allele is correlated with lower expression of IGF2 isoform 2 (as measured by expression levels of the exon 1-2 junction) in liver (GG, n = 21; GA, n = 9; AA, n = 4) (A) and in adipose tissue (GG, n = 83; GA, n = 43; AA, n = 5) (B).

Figure 4

Expression of IGF2 isoform 2 is associated with T2D and HbA_1c. A: Boxplots representing the expression of IGF2 isoform 2 across T2D case and control subjects in individuals homozygous for the G common allele. The linear model P value represents the association between IGF2 isoform 2 expression, adjusted by age, BMI, and sex. B: IGF2 isoform 2 positively correlates with higher HbA_1c in participants without diabetes. The gray area limited by the dashed red lines represents the 95% CI of the slope of the linear regression. Patients with HbA_1c above 6.5% did not have T2D according to the diagnostic criteria of Mexico at the time of extraction, as HbA_1c was not considered a criterion in Mexico at the time of extraction. Therefore, none of the subjects were receiving any glucose-lowering treatment. For clarity, as the genotype is strongly associated with IGF2 isoform 2 expression, only individuals carrying the GG genotype are plotted in A and B.

Figure 5

Phenome-wide analysis of rs149483638 variant. The protective variant was tested for association across 18 different disease traits previously categorized in the subsample of GERA cohort of Latino ancestry (5,896 individuals). Although the rs149483638 variant was associated with reduced risk of T2D, there was no significant association seen for other 18 conditions. Association analyses were done by logistic regression analyses, considering additive model, and correcting for age, BMI, sex, and the first two principal components to correct for population stratification. IBS, irritable bowel syndrome; Mac. Degen., macular degeneration; Psychiatric, any psychiatric condition; PVD, peripheral vascular disease; Stress, acute reaction to stress.