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. 2023 May 30;3(7):100339.
doi: 10.1016/j.xgen.2023.100339. eCollection 2023 Jul 12.

Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins

Natalie DeForest  1   2 , Babu Kavitha  3 , Siqi Hu  1 , Roi Isaac  1 , Lynne Krohn  4 , Minxian Wang  5 , Xiaomi Du  1 , Camila De Arruda Saldanha  1 , Jenny Gylys  1 , Edoardo Merli  1 , Ruben Abagyan  6 , Laeya Najmi  5   7 , Viswanathan Mohan  8 ; Alnylam Human Genetics; AMP-T2D Consortium; Jason Flannick  5   9 , Gina M Peloso  10 , Philip L S M Gordts  1   11 , Sven Heinz  1 , Aimee M Deaton  4 , Amit V Khera  5   12 , Jerrold Olefsky  1 , Venkatesan Radha  3 , Amit R Majithia  1
Affiliations

Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins

Natalie DeForest et al. Cell Genom. .

Abstract

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.

Keywords: ANGPTL3; HNF1A; PCSK9; UK Biobank; atherosclerosis; coronary artery disease; deep mutational scan; diabetes; gain of function; inflammation; saturation mutagenesis.

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Conflict of interest statement

A.M.D. and L.K. are employees and shareholders of Alnylam Pharmaceuticals.

Figures

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Graphical abstract
Figure 1
Figure 1
Identification of GOF variants from comprehensive functional testing of 11,970 HNF1A variants in human hepatocytes (A) A library of 11,970 HNF1A constructs was synthesized, with each construct encoding a single amino acid substitution. The construct library was introduced into HUH7 hepatocytes (deleted for endogenous HNF1A) at a dilution of one construct per cell. The resulting polyclonal population of HUH7 hepatocytes was separated via FACS according to the expression of the known HNF1A transcriptional _target TM4SF4 and sorted into low (−) and high (+) bins of HNF1A activity. Activity cutoffs were established through flow cytometry experiments of HNF1A KO cells (dashed red line) and WT cells (dashed green line). Each bin of cells was sequenced at the transgenic HNF1A locus to identify and tabulate the introduced variants. Each HNF1A variant was assigned a function score based on its abundance in the low and high TM4SF4 expression bins. (B) Heatmap of 11,970 HNF1A variant function scores, arranged according to the primary amino acid sequence (rows). Function scores lower than WT are shaded red, and function scores greater than WT are shaded blue. Function scores averaged (mean) at each amino acid position are plotted to the right, showing the level of tolerance for any amino acid substitution away from WT at each position. (C) Mutation tolerance scores as described in (B) overlaid on the crystalized protein structure of HNF1A DNA-binding domain (PF04814). Positions intolerant of amino acid changes (i.e., lower function scores) are shaded red. Helices that make direct contacts with the DNA are the most intolerant of mutations. (D) (Left panel) HNF1A function scores ranked for all 11,970 amino acid variants tested, and ClinVar-annotated pathogenic variants (n = 29) are highlighted in red. (Right panel) Function bins correspond to variants with function scores above (GOF), within (neutral), or below (LOF) ± 1 Z-score of the synonymous distribution, shown with the total number of variants per bin. Overlaid are the function score distributions of the 613 synonymous HNF1A variants tested (purple) and the 29 ClinVar pathogenic variants (red).
Figure 2
Figure 2
GOF HNF1A variants are carried in the general population and increase transcriptional activity in multiple cellular contexts (A) (Left panel) Selected human HNF1A variants with function scores greater than WT highlighted in blue among the full distribution of all 11,970 amino acid variants tested. The shaded purple box represents ± 1 Z-score of the distribution of 613 synonymous HNF1A variants tested. (Right panel) The distributions of function scores of rare protein-coding HNF1A variants (n = 444) identified from 454,756 sequenced individuals in the UK Biobank (UKB) and of the 613 synonymous variants. An upper tail of function-increasing variants in UKB individuals is highlighted in blue (n = 94 unique variants in 1,469 individuals). (B) Location of top-scoring variants selected for validation along the HNF1A protein primary amino acid sequence with number of human variant carriers identified in 454,756 exome sequences from the UKB. (C) Putative GOF variants were individually recreated and tested for transcriptional activation using luciferase reporters in HeLa cells (which lack endogenous HNF1A activity) on the rat albumin promoter. Activity measurements are shown as percentages of WT HNF1A activity ± SEM; n = 3. Basal promoter activity in cells is measured by transfection of vectors lacking the HNF1A transgene (empty) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, linear mixed model). (D) The same variants described in (B) were tested for transcriptional activity in mouse insulinoma MIN6 cells on the HNF4A-P2 promoter. (E) GOF HNF1A variant constructs were transfected into HNF1A-deleted HUH7 hepatocytes, and ANGPTL3 levels were measured by ELISA 48 h post-confluency (n = 3 per variant). Transfection with HNF1A p.L348F, p.T196A, and p.T515M increases ANGPTL3 secretion compared with WT (∗∗∗p < 0.0005,∗∗p < 0.005, ∗p < 0.05, linear mixed model).
Figure 3
Figure 3
HNF1A GOF variant carriers in the population are protected from T2D but show elevated levels of serum atherogenic risk factors Shape and fill indicate the ancestry and T2D status, respectively, of individuals included in each analysis. All phenotypes are adjusted for age, age2, sex, and the first 10 principal components of ancestry. T2D, type 2 diabetes; SD, standard deviation; LOF, loss of function; GOF, gain of function; CRP, C-reactive protein; LDL, low-density lipoprotein cholesterol; ApoB, apolipoprotein B; GGT, gamma-glutamyl transferase; TC, total cholesterol; TG, triglycerides; ApoA-I, apolipoprotein A-I; HDL, high-density lipoprotein cholesterol; HbA1c, glycated hemoglobin. (A) Association of HNF1A function score and T2D risk using logistic regression in the UKB HNF1A rare protein-coding variant carriers identified among the 454,756 exome-sequenced individuals. Odds ratios and the 95% confidence interval are shown. (B) Association of LOF and GOF HNF1A variants with T2D in the UKB and AMP-T2D (n = 20,791 cases, 24,440 controls) using logistic regression. Odds ratios and the 95% confidence interval are shown, and fixed effects inverse-variance meta-analysis was used to combine the results. (C) Association of functional HNF1A variants with cardiovascular disease risk factors in the UKB rare variant carriers (European n = 3,089, multi-ancestry n = 4,302, linear mixed-effects regression). Effect size and the 95% confidence interval are shown for each phenotype. (D) Association of liver-specific predicted HNF1A gene expression with disease risk factors tested in (C) among all UKB European genotyped individuals (n = 407,227, filled squares) and non-T2D European genotyped individuals (n = 376,043, open squares).
Figure 4
Figure 4
HNF1A transcriptionally regulates complement activation, coagulation, and pro-atherogenic gene expression programs in hepatocytes (A) Differentially expressed genes between WT and HNF1A KO HUH7 and Hep3B cell lines quantified by mRNA sequencing (n = 3 per group). The dotted red line indicates an adjusted p value threshold of 0.05, with genes falling above the line meeting a global significance threshold for differential expression. (B) Gene set enrichment analysis (GSEA) was performed on mRNA sequencing data shown in (A) to identify pathways altered by deletion of HNF1A (HNF1A KO vs. WT). Significant genesets in at least one cell line are shown. The effect of GOF in HNF1A on these pathways was examined by complementing HNF1A KO HUH7 cells with WT HNF1A and the GOF p.T196A variant (n = 3 per group), followed by mRNA sequencing, differential expression, and GSEA (HNF1A p.T196A vs. WT). Downregulated pathways (negative normalized enrichment scores) are shaded yellow, and upregulated pathways are shaded blue. Size represents the -log10(p value), and significantly enriched pathways (p < 0.05) are outlined in red. (C) HNF1A was reintroduced via doxycycline-inducible transgenes into HNF1A KO hepatocyte cell lines and global gene expression was measured (TPM, transcripts per million; n = 3 per group). In a dose-dependent fashion, HNF1A regulates genes involved in oxidative stress and lipid regulation pathways. Significance levels from regressing TPM on HNF1A levels are shown for each gene and cell type if significant (∗∗∗p < 10−6, ∗∗p < 10−3, ∗p < 0.05, linear model). GGT1, gamma glutamyltransferase 1; PCSK9, proprotein convertase subtilisin/kexin type 9; APOA1, apolipoprotein A1; APOB, apolipoprotein B.
Figure 5
Figure 5
Graphical summary of proposed mechanism through which GOF in HNF1A decreases diabetes risk through pancreas-specific mechanisms but increases the hepatic secretion of atherogenic risk factors Carriers of GOF variants in HNF1A have reduced T2D risk and HbA1c through enhanced beta cell function but have increases in CRP and GGT and present a pro-atherogenic profile (suppression of ApoA-I/HDL levels and increased hepatic secretion of ANGPTL3 and ApoB/LDL levels through increased transcription of PCSK9). Red arrows indicate metabolically harmful outcomes, and blue arrows represent metabolically beneficial outcomes. GOF, gain of function; CRP, C-reactive protein; GGT, gamma-glutamyl transferase; ApoA-I, apolipoprotein A-I; HDL, high-density lipoprotein cholesterol; ANGPTL3, angiopoietin like 3; TG, triglycerides; PCSK9, proprotein convertase subtilisin/kexin type 9; ApoB, apolipoprotein B; LDL, low-density lipoprotein cholesterol; HbA1c, glycated hemoglobin.
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