Loss-of-function mutations in SLC30A8 protect against type 2 diabetes

Abstract

Loss-of-function mutations protective against human disease provide in vivo validation of therapeutic targets, but none have yet been described for type 2 diabetes (T2D). Through sequencing or genotyping of ~150,000 individuals across 5 ancestry groups, we identified 12 rare protein-truncating variants in SLC30A8, which encodes an islet zinc transporter (ZnT8) and harbors a common variant (p.Trp325Arg) associated with T2D risk and glucose and proinsulin levels. Collectively, carriers of protein-truncating variants had 65% reduced T2D risk (P = 1.7 × 10(-6)), and non-diabetic Icelandic carriers of a frameshift variant (p.Lys34Serfs*50) demonstrated reduced glucose levels (-0.17 s.d., P = 4.6 × 10(-4)). The two most common protein-truncating variants (p.Arg138* and p.Lys34Serfs*50) individually associate with T2D protection and encode unstable ZnT8 proteins. Previous functional study of SLC30A8 suggested that reduced zinc transport increases T2D risk, and phenotypic heterogeneity was observed in mouse Slc30a8 knockouts. In contrast, loss-of-function mutations in humans provide strong evidence that SLC30A8 haploinsufficiency protects against T2D, suggesting ZnT8 inhibition as a therapeutic strategy in T2D prevention.

Figure 1. Over-expression of p.Arg138X- and p. Ser34fsX50-ZnT8 in HeLa cells

We sought to experimentally evaluate whether the p.Arg138X or p. Ser34fsX50 ZnT8 variants resulted in decreased ZnT8 expression and/or activity. (a) Depiction of SLC30A8 open reading frames in C-terminal V5-tagged constructs (tag highlighted in green). (b) Western blot of HeLa lysates following transient over-expression of V5-tagged ZnT8 variants (anti-V5-tag). Antibody against tubulin was used as a loading control for each sample, and untransfected cell lysate was used to demonstrate specificity of anti-V5 antibody. (c, d) Immunofluorescent staining of ZnT8 variant expression in (c) HeLa and (d) Ins1e cells. ZnT8 was detected using antibodies against the C-terminal V5-tag (anti-V5) or the N-terminus of the endogenous protein (anti-ZnT8), as indicated. BFP-V5 and untransfected HeLa cells serve as controls. Cells were co-stained with Hoechst-33342 to mark nuclei. Within each row of images for the indicated antibody and objective, identical exposure times were used across all proteins. (e) ZnT8 variant expression, as detected by anti-V5 immunostaining, following 4hr treatment with inhibitors of the lysosome (chloroquine, 100 μM) or the proteasome (MG132, 10 μM). Images were acquired using a 10x objective and identical exposure times. Scale bars, 100 μM.

Figure 2. Protein-truncating variants identified in SLC30A8

Through sequencing and genotyping of nearly 150,000 individuals across 5 ethnicities, we identified 12 SLC30A8 variants – each rare and predicted to cause premature protein truncation. (a) Shown is the position of each variant on the islet-specific SLC30A8 transcript (NM_173851). p.Met50Ile is predicted to alter the initiator codon in other transcripts of SLC30A8. Lines are drawn from each variant to ethnicities for which carriers were observed, with greater widths corresponding to ethnicities with more observations. Lines are further drawn from each ethnicity to the populations (cohorts) from which carriers were identified. From left, cohorts are: JHS, WFS, Botnia, Danish, deCODE, Finnish, HUNT2, KORA, Malmo, PIVUS/ULSAM, WTCCC, LOLIPOP, Singapore Indians, and KARE (cohort information in Supplementary Information). Ethnicities or cohorts with no observations are not shown. (b) Graphical representation of the case and control frequencies for each observed variant; case frequencies in red (above) and control frequencies in blue (below). Wider bars correspond to variants with more observations. A quantitative and complete representation of these data is given in Table 1.