University of Southern Denmark Loss-of-function variants in ADCY 3 increase risk of obesity and type 2 diabetes

We have identified a variant in adenylate cyclase 3 (ADCY3) associated with markedly increased risk of obesity and type 2 diabetes in the Greenlandic population. The variant disrupts a spliceacceptor site and carriers display decreased ADCY3 RNA expression. Additionally, we observe an enrichment of rare ADCY3 loss-of-function variants among type 2 diabetes patients in trans-ethnic Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Corresponding authors: Torben Hansen, torben.hansen@sund.ku.dk, Anders Albrechtsen, albrecht@binf.ku.dk, Marit E. Jørgensen, marit.eika.joergensen@regionh.dk. URLs Accelerating Medicines Partnership Type 2 Diabetes Knowledge Portal: http://www.type2diabetesgenetics.org/ Genome Aggregation Database (gnomAD): http://gnomad.broadinstitute.org/ GTEx Portal: https://www.gtexportal.org/ FastQC: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Contributions T.H. and A.A. conceived and headed the project. I.M. and A.A. designed the statistical setup for the association testing, while T.H., N.G, M.K.A. and O.P. designed the experimental setup for the DNA extraction, genotyping and sequencing. M.E.J. and P.B. were PIs of the population studies in Greenland, and together with C.V.L.L., and I.K.D.-P. they provided the Greenlandic samples, collected and defined the phenotypes and provided context of these samples. I.M., N.G., E.J. and A.A. performed the association analyses. T.K. and Y.M. designed the experimental setup for RNA extraction and sequencing. A.G, D.S, G.D. and E.Z. performed the loss-of-function analysis in the Greek cohorts. K.V.S., M.D. and R.A. performed the RNA sequence analysis. N.G., I.M., M.K.A., A.A. and T.H. wrote the majority of the manuscript with input from all authors. All authors approved the final version of the manuscript. Competing Financial Interests Statement The authors report no competing financial interests. Europe PMC Funders Group Author Manuscript Nat Genet. Author manuscript; available in PMC 2018 July 08. Published in final edited form as: Nat Genet. 2018 February ; 50(2): 172–174. doi:10.1038/s41588-017-0022-7. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts cohorts. These findings provide novel information on disease etiology relevant for future treatment strategies. Identification of homozygous loss-of-function mutations in humans may readily inform about the biological impact of specific genes and point to novel drug targets. We previously identified a loss-of-function variant in TBC1D4 segregating at high frequency in the Greenlandic population displaying a high impact on risk of type 2 diabetes1, confirming the advantage of studying the Greenlandic population due to its extreme demographic history2. Motivated by this, we screened for novel loss-of-function variants in the exome sequencing data from 27 individuals in nine trios that were used to identify the causal TBC1D4 loss-offunction variant1. We identified 46 such variants (Supplementary Table 1) and intersected the location of these variants with loci known to associate with obesity or body mass index (BMI) (Supplementary Fig. 1). One of the variants, which was present in one copy in one of the trio's parents, was situated in a locus where a common non-coding variant has been shown to be associated with BMI in adults and children in genome-wide association studies (GWAS)3,4. The variant (hg19: 2-25050478-C-T, c.2433-1G>A) is predicted to destroy a splice-acceptor site in exon 14 (NM_004036.4) (Fig. 1A) of ADCY3. For this reason, we investigated the specific variant further by genotyping it in two Greenlandic cohorts. This revealed an overall minor allele frequency of 2.3% in the Greenlandic study population (N=4,038, N-heterozygous=172, N-homozygous=7), and a frequency of 3.1% in the Inuit ancestry part of the population. Importantly, the seven homozygous carriers had a 7.3 kg/m2 higher BMI (P=0.00094) compared with the remaining study population (Table 1). Interestingly, we also observed that three of the seven homozygous carriers had type 2 diabetes (P=7.8×10-5, Table 1), while one had impaired fasting glucose and one impaired glucose tolerance. Notably, the association with type 2 diabetes remained significant after adjustment for BMI (P=6.5×10-4), suggesting it is not simply mediated by increased BMI. The effects on BMI and type 2 diabetes were also observed, although with smaller effect sizes, when data were analyzed according to an additive genetic model (Table 1). However, when we compared the recessive and additive models with the full genotype model, we rejected the additive model (BMI: P=0.002; type 2 diabetes: P=0.004), but not the recessive model (BMI: P=0.17; type 2 diabetes: P=0.095). This suggests that the recessive model is appropriate for explaining the effect of the c.2433-1G>A variant. To further characterize homozygous ADCY3 c.2433-1G>A carriers in the Greenlandic cohorts, we analyzed a number of additional traits related to BMI and type 2 diabetes. The homozygous carriers had an 8.1 percentage points higher body fat percentage (P=0.0024) and a 17 cm larger waist circumference (P=0.0017). In addition, the homozygous carriers had nominally higher levels of fasting and 2-h plasma glucose after an oral glucose tolerance test (P=0.022 and P=0.035, respectively; Table 1). Finally, we also observed nominally significant effects on dyslipidemia and insulin resistance (Supplementary Table 2). The c.2433-1G>A ADCY3 variant was not observed in sequencing data of up to 138,000 individuals from non-Greenlandic populations collected by the Genome Aggregation Database Consortium5 (gnomAD). To generalize our findings to other populations, we therefore investigated the effect of loss-of-function variants in ADCY3 more generally by Grarup et al. Page 2 Nat Genet. Author manuscript; available in PMC 2018 July 08. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts analyzing 18,176 samples with exome sequence data generated by the GoT2D, T2DGenes, SIGMA and LuCAMP consortia6,7,8, which are available at the Accelerating Medicines Partnership Type 2 Diabetes Knowledge Portal (AMP-T2D). No homozygous ADCY3 lossof-function carriers were observed in this dataset, but the analysis included seven predicted ADCY3 loss-of-function variants (Fig. 1A) observed in the heterozygous form in eight individuals, and we observed an enrichment of carriers among type 2 diabetes cases compared with non-diabetic controls (7/8,845 in cases, 1/9,323 in controls, OR 8.6, P=0.044, Supplementary Table 3-5). To further substantiate these findings, exome sequence data from 9,928 Finnish individuals from the METSIM cohort9 were screened for loss-offunction variants in ADCY3; however, none were identified (Markku Laakso, personal communication). Furthermore, we did not find any loss-of-function ADCY3 variants in whole genome sequence data for 3,124 individuals from two Greek isolated populations as part of the HELIC study10. Finally, in up to 138,000 individuals in the gnomAD data, 48 predicted loss-of-function variants were found. All variants had minor allele frequency below 0.007% and only a single homozygous loss-of-function carrier was found for the African-specific ADCY3 c.1072-1G>A variant. Since we cannot obtain phenotypic data for the gnomAD samples, we are unable to evaluate the impact of these variants on obesity and type 2 diabetes. To investigate the functional impact of the ADCY3 c.2433-1G>A variant, we performed deep RNA sequencing in leukocytes from 17 Greenlandic individuals (7 GG carriers, 6 GA carriers and 4 AA carriers). The RNA sequencing data confirmed that ADCY3 was expressed in the wild type GG carriers and that exon 14 (NM_004036.4) of ADCY3 was expressed and spliced in its canonical form (Supplementary Fig. 2A). The inclusion of exon 14 in the mature mRNA was further confirmed by RNA sequence data from adipose tissue of a healthy Caucasian female donor11. Importantly, we found that the overall RNA expression level of ADCY3 was severely decreased in the homozygous AA carriers, while the heterozygous GA carriers showed an intermediate expression level (Fig. 1B). The RNA sequencing data further confirmed that the predicted disruption of exon 14 splice-acceptor site by variant c.2433-1G>A has an impact on the molecular phenotype. Specifically, the data predict that two novel ADCY3 isoforms are expressed in the variant carriers: one transcript isoform where exon 14 is skipped and an alternative splice-acceptor site at exon 15 is used and one transcript isoform in which the intron between exon 13 and exon 14 is retained (Supplementary Fig. 2B). We quantified these alternative splicing events by comparing the expression of the three predicted ADCY3 isoforms using isoform fraction (IF) (Fig. 1C) and the percentage of spliced in (PSI) at the relevant splice sites (Supplementary Fig. 2B). Both analyses demonstrated that the homozygous AA carriers had severely affected splice pattern and mainly used intron retention (median IF 0.38, median PSI 75%) or exon skipping (IF 0.53, PSI 24%), while the wild type GG carriers had the canonical splice pattern (IF 0.88, PSI 87%). In all analyses, heterozygous carriers showed an intermediate level of alternative splicing (Fig. 1C; Supplementary Fig. 2B & C). Importantly, we predict the isoform with an intron retention to be sensitive to nonsense mediated decay due to a premature stop codon12 (Fig. 1A). This predicted degradation naturally would lead to further reduction of ADCY3 protein levels. Grarup et al. Page 3 Nat Genet. Author manuscript; available in PMC 2018 July 08. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts ADCY3 encodes an adenylate cyclase with a wide tissue distribution showing high l