Individual risk of type 2 diabetes (T2D) is modified by perturbations of adipose mass, distribution and function. To investigate mechanisms responsible, we explored the molecular, cellular, and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression, and modulate, in trans, expression of 385 genes. We demonstrate that, in human cellular studies, reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and, in mice, adipose-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that KLF14 T2D risk-allele carriers shift body fat from gynoid to abdominal stores, and display a marked increase in adipocyte cell size: these effects on fat distribution, and the T2D-association, are female-specific. Metabolic risk associated with variation at this imprinted locus depends on both the sex of the subject, and of the parent from whom the risk-allele derives.

Summary Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a novel regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss-of-function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2 deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

SUMMARY Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project’s human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.

Objective Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology—permanent alteration of genomic DNA sequences—mandates that it be tested in vivo against human genes in normal human cells with human genomes in order to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. Approach and Results We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9) genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. Conclusions This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.