HNF1A Variants Research Articles

Rare variant analyses in 51,256 type 2 diabetes cases and 370,487 controls reveal the pathogenicity spectrum of monogenic diabetes genes

Type 2 diabetes (T2D) genome-wide association studies (GWASs) often overlook rare variants as a result of previous imputation panels’ limitations and scarce whole-genome sequencing (WGS) data. We used TOPMed imputation and WGS to conduct the largest T2D GWAS meta-analysis involving 51,256 cases of T2D and 370,487 controls, targeting variants with a minor allele frequency as low as 5 × 10−5. We identified 12 new variants, including a rare African/African American-enriched enhancer variant near the LEP gene (rs147287548), associated with fourfold increased T2D risk. We also identified a rare missense variant in HNF4A (p.Arg114Trp), associated with eightfold increased T2D risk, previously reported in maturity-onset diabetes of the young with reduced penetrance, but observed here in a T2D GWAS. We further leveraged these data to analyze 1,634 ClinVar variants in 22 genes related to monogenic diabetes, identifying two additional rare variants in HNF1A and GCK associated with fivefold and eightfold increased T2D risk, respectively, the effects of which were modified by the individual’s polygenic risk score. For 21% of the variants with conflicting interpretations or uncertain significance in ClinVar, we provided support of being benign based on their lack of association with T2D. Our work provides a framework for using rare variant GWASs to identify large-effect variants and assess variant pathogenicity in monogenic diabetes genes.

Genomic and Transcriptomic Profile of HNF1A-Mutated Liver Adenomas Highlights Molecular Signature and Potential Therapeutic Implications.

Hepatocellular adenomas (HAs) are tumors that can develop under different conditions, including in patients harboring a germline mutation in HNF1A. However, little is known about the pathogenesis of such disease. This work aims to better define what mechanisms lie under the development of this condition. Six HAs were sampled from the liver of a 17-year-old male affected by diabetes and multiple hepatic adenomatosis harboring the heterozygous pathogenic germline variant c.815G>A, p.(Arg272His) in HNF1A, which has a dominant negative effect. All HAs were molecularly characterized. Four of them were shown to harbor a second somatic HNF1A variant and one had a mutation in the ARID1A gene, while no additional somatic changes were found in the remaining HA and normal parenchyma. A transcriptomic profile of the same HA samples was also performed. HNF1A biallelic mutations were associated with the up-regulation of several pathways including the tricarboxylic acid cycle, the metabolism of fatty acids, and mTOR signaling while angiogenesis, endothelial and vascular proliferation, cell migration/adhesion, and immune response were down-regulated. Contrariwise, in the tumor harboring the ARID1A variant, angiogenesis was up-modulated while fatty acid metabolism was down-modulated. Histological analyses confirmed the molecular data. Independently of the second mutation, energetic processes and cholesterol metabolism were up-modulated, while the immune response was down-modulated. This work provides a complete molecular signature of HNF1A-associated HAs, analyzing the association between specific HNF1A variants and the development of HA while identifying potential new therapeutic targets for non-surgical treatment.

Diazoxide hypersensitivity in neonatal hyperinsulinism due to HNF4A variants

Diazoxide hypersensitivity in neonatal hyperinsulinism due to HNF4A variants

Our Experiences and Learnings in Diagnosing MODY from Non-Institutional-Based Diabetes Care Clinics

Abstract Introduction: Maturity-onset diabetes of the young (MODY) is a rare group of disorders characterised by impaired functions or development of pancreatic islets and monogenic diabetes at a young age. Diagnosing MODY can be rewarding for both clinicians and patients as it can change the management from generic to targeted therapy. Methods: This study reports the retrospective analysis of data collected from four clinics between March 2016 and February 2023 from Lucknow, a city in northern India. Fifty-three individuals are suspected to be affected by MODY based on ISPAD guidelines. Following a detailed clinical evaluation, they were referred for genetic diagnostic testing. Results: The cohort consists of 19 females and 34 males with a mean age of diagnosis of 25.3 years and a body mass index of 22.3 Kg/m2. Genetic testing detected variants in 13/53 (~24.5%) individuals. Five cases had significant pathogenic/likely pathogenic variants, HNF1A gene in two [(p.Phe268LeufsTer74) (p.Arg200Gln)], one each in HNF4A (Arg311His), PDX1(p.Ala228GlyfsTer33), and a case with suggestive digenic variants in HNF1A gene (p.Arg200Gln) and HNF1B [(p.Leu13Met)]. Variants of uncertain significance (VUSs) with inconclusive evidence of pathogenicity were reported in eight patients, and five were considered to be clinically significant as they are lean young onset, sulfonylurea-responsive, and presented with diabetes without acanthosis nigricans and with high pretest probability. These individuals harboured variants in HNF1A (p.Thr425_Thr429delinsPro), HNF1B (p.Ser19Phe), CEL (p.Val681ArgfsTer6), ABCC8 (p.Ile872Met), and KCNJ11 (p.Arg221Cys) genes. Conclusion: We found a diagnostic yield of around 10% of pathogenic or likely pathogenic variants in individuals who were suspected to have MODY. As it is a field that is still evolving, we might consider starting with oral agents under close supervision in those individuals who have VUS; there are some proportions of individuals who might not have classical sulfonylurea-responsive genetic variants, but they might respond to it.

Low C reactive protein-alleles in hepatocyte nuclear factor 1A are associated with an increased risk of cardiovascular disease: a Meta-analysis.

Rare variants in HNF1A cause both maturity onset diabetes of the young 3 (HNF1A-MODY) and reduced serum C reactive protein (CRP) levels. Common variants of HNF1A are associated with serum CRP and type 2 diabetes, but inconsistently with cardiovascular disease (CVD). Our study aimed to investigate the association of low CRP-alleles in HNF1A with CVD and indirectly evaluate the CVD risk of HNF1A-MODY patients because of unavailability of enough cases to study their clinical outcomes. A literature search was performed using PubMed, Embase and Cochrane Library databases from inception to December 2023. All relevant studies concerning the association of HNF1A with CRP, CVD, lipid and type 2 diabetes were included. Odds ratios (ORs), 95% confidence intervals (CIs) and study characteristics were extracted. Three common coding variants of HNF1A (rs1169288, rs2464196, rs1169289) were examined. The minor alleles of these variants correlated with low CRP levels (OR 0.89, 95%Cl 0.86-0.91; OR 0.89, 95%Cl 0.88-0.91; OR 0.89, 95%Cl 0.88-0.91, respectively). Their low CRP-alleles were associated with increased risk of CVD (OR 1.03, 95%CI 1.03-1.04), higher LDL-cholesterol levels (OR 1.07, 95%CI 1.04-1.10) and elevated risk of type 2 diabetes (OR 1.04, 95%CI 1.01-1.08). Our study revealed an association between low CRP-alleles in HNF1A and a high CVD risk, which indicated that anti-diabetic drugs with cardiovascular benefits such as GLP-1 receptor agonist should be recommended as first-line choice for HNF1A-MODY.

Chinese carrier of the HNF1A p.Gln444fs variant exhibits enhanced response to sulfonylureas

BackgroundWe assessed the response to sulfonylureas and the functional characteristics of HNF1A mutations in patients with maturity-onset diabetes of the young type 3 (MODY3). MethodsWe recruited a family with suspected MODY in this study, and gene sequencing (whole-exome sequencing) was used to screen germline mutations. Luciferase reporter assays were used to evaluate the activity of the mutated genes. ResultsHeterozygous HNF1A variant (NM_000545.8:c.1330_1331del, p.Gln444fs) was identified in the proband and was not found in his father, grandmother, and nonrelated healthy controls. The mutant protein had 552 amino acids, 110 fewer than the wild type protein. Furthermore, the amino acid sequence was completely different between the mutant protein and the wild type protein starting from the 444th amino acid. Luciferase reporter assays revealed that the variant had impaired HNF4A promoter–regulation activity. The patient did not achieve good hypoglycemic effects during long-term treatment with insulin and metformin. The effect of hypoglycemic treatment was highly significant after the addition of sulfonylurea drugs. ConclusionsThe HNF1A p.Gln444fs variant associated with MODY3, and most likely a truncated protein, impaired HNF1A transcriptional activity. The variant carrier experienced an enhanced response to sulfonylureas.

HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known (ACY3, HAAO, HNF1A, MAP3K11) and previously unidentified (ABCD3, CDKN2AIP, USH1C, VIL1) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1, GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation.

Genetic insights into fetal kidney development: Variants in HNF1A and PKHD1 genes

The orchestration of fetal kidney development involves the precise control of numerous genes, including HNF1A, HNF1B and PKHD1. Understanding the genetic factors influencing fetal kidney development is essential for unraveling the complexities of renal disorders. This study aimed to search for disease-causing variants in HNF1A, HNF1B, PKHD1 genes, among fetus and babies or via parental samples, using sanger sequencing, NGS technologie and MLPA.The study revealed an absence of gene deletions and disease-causing variants in the HNF1B gene. However, five previously SNPs in the HNF1A gene were identified in four patients (patients 1, 2, 3, and 4). These include c.51C > G (Exon1, p. Leu17=), c.79A > C (Exon1, p. Ile27Leu), c.1375C > T (Exon7, p. Leu459=), c.1460G > A (Exon7, p. Ser487Asn), and c.1501 + 7G > A (Intron7). Additionally, in addition to previously SNPs identified, a de novo heterozygous missense mutation (p.E508K) was detected in patient 4. Furthermore, a heterozygous mutation in exon 16 (p. Arg494*; c.1480C > T) was identified in both parents of patient 5, allowing predictions of fetal homozygosity.Bioinformatic analyses predicted the effects of the c.1522G > A mutation (p.E508K) on splicing processes, pre-mRNA structures, and protein instability and conformation. Similarly, the c.1480C > T mutation (p. Arg494*) was predicted to introduce a premature codon stop, leads to the production of a shorter protein with altered or impaired function.Identification of variants in the HNF1A and in PKHD1 genes provides valuable insights into the genetic landscape of renal abnormalities in affected patients. These findings underscore the heterogeneity of genetic variants contributing to renal disorders and emphasize the importance of genetic screening.

Functional Analysis of a Novel HNF4A Variant Identified in a Patient With MODY1.

HNF4A-maturity-onset diabetes of the young (MODY1) is a relatively rare subtype of monogenic diabetes caused by loss of function of the HNF4A gene, which encodes the transcription factor HNF4α. HNF4α is known to form heterodimers, and the various combinations of isoforms that make up these heterodimers have been reported to result in a diversity of targeted genes. However, the function of individual HNF4α variant isoforms and the heterodimers comprising both wild-type (WT) and variant HNF4α have not yet been assessed. In this study, we analyzed the functional consequence of the HNF4A D248Y variant in vitro. We investigated the case of a 12-year-old Japanese girl who developed diabetes at age 11 years. Genetic sequencing detected a novel heterozygous missense HNF4A variant (c.742G > T, p.Asp248Tyr; referred as "D248Y") in the patient and her relatives who presented with diabetes. Although the WT HNF4α isoforms (HNF4α2, HNF4α3, HNF4α8, HNF4α9) enhanced the INS gene promoter activity in HepG2 cells, the promoter activity of D248Y was consistently low across all isoforms. The presence of D248Y in homodimers and heterodimers, comprising either HNF4α8 or HNF4α3 or a combination of both isoforms, also reduced the INS promoter activity in Panc-1 cells. We report the clinical course of a patient with HNF4A-MODY and the functional analysis of novel HNF4A variants, with a focus on the isoforms and heterodimers they form. Our results serve to improve the understanding of the dominant-negative effects of pathogenic HNF4A variants.

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and β-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

Dominant-negative HNF1α mutant promotes liver steatosis and inflammation by regulating hepatic complement factor D

Patients with HNF1A variants may develop liver steatosis, while the underlying mechanism is still unclear. Here, we established a mouse model carrying the dominant-negative HNF1α P291fsinsC mutation (hHNF1Amut/-) and found that the mutant mice developed liver steatosis spontaneously under the normal chow diet. Transcriptome analysis showed significant upregulation of Cfd and other genes related to innate immune response in the liver of hHNF1Amut/- mice. The changes in lipid metabolism and complement pathways were also confirmed by proteomics. We demonstrated that HNF1α inhibited CFD expression in hepatocytes, and the P291fsinsC mutant could reverse this inhibitory effect. Furthermore, the suppression of CFD with specific inhibitor or siRNAs reduced triglyceride levels in hepatocytes, suggesting that CFD regulated hepatocyte lipid deposition. Our results demonstrate that the HNF1α P291fsinsC mutant promotes hepatic steatosis and inflammation by upregulating CFD expression, and targeting CFD may delay the progression of nonalcoholic fatty liver disease.

Evaluation in Monogenic Diabetes of the Impact of GCK, HNF1A, and HNF4A Variants on Splicing through the Combined Use of In Silico Tools and Minigene Assays

Variants in GCK, HNF1A, and HNF4A genes are the three main causes of monogenic diabetes. Determining the molecular etiology is essential for patients with monogenic diabetes to benefit from the most appropriate treatment. The increasing number of variants of unknown significance (VUS) is a major issue in genetic diagnosis, and assessing the impact of variants on RNA splicing is challenging, particularly for genes expressed in tissues not easily accessible as in monogenic diabetes. The in vitro functional splicing assay based on a minigene construct is an appropriate approach. Here, we performed in silico analysis using SpliceAI and SPiP and prioritized 36 spliceogenic variants in GCK, HNF1A, and HNF4A. Predictions were secondarily compared with Pangolin and AbSplice-DNA bioinformatics tools which include tissue-specific annotations. We assessed the effect of selected variants on RNA splicing using minigene assays. These assays validated splicing defects for 33 out of 36 spliceogenic variants consisting of exon skipping (15%), exonic deletions (18%), intronic retentions (24%), and complex splicing patterns (42%). This provided additional evidence to reclassify 23 out of 31 (74%) VUS including missense, synonymous, and intronic noncanonical splice site variants as likely pathogenic variants. Comparison of in silico analysis with minigene results showed the robustness of bioinformatics tools to prioritize spliceogenic variants, but revealed inconsistencies in the location of cryptic splice sites underlying the importance of confirming predicted splicing alterations with functional splicing assays. Our study underlines the feasibility and the benefits of implementing minigene-splicing assays in the genetic testing of monogenic diabetes after a prior in-depth in silico analysis.

High-fat diet plus HNF1A variant promotes polyps by activating β-catenin in early-onset colorectal cancer.

The incidence of early-onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here, we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 α (HNF1AA98V, rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9, and the mice were placed on either a high-fat diet (HFD) or high-sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however, 19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes, and Wnt/β-catenin signaling components in the HNF1A mutant relative to the WT mice. Mouse polyps and colon cancers from participants carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared with WT HNF1A. Collectively, our study shows that the HNF1AA98V variant plus a HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.

Role of HNFA1 Gene Variants in Pancreatic Beta Cells Function and Glycaemic Control in Young Individuals with Type 1 Diabetes.

The HNF1A transcription factor, implicated in the regulation of pancreatic beta cells, as well as in glucose and lipid metabolism, is responsible for type 3 maturity-onset diabetes of the young (MODY3). HNF1A is also involved in increased susceptibility to polygenic forms of diabetes, such as type 2 diabetes (T2D) and gestational diabetes (GD), while its possible role in type 1 diabetes (T1D) is not known. In this study, 277 children and adolescents with T1D and 140 healthy controls were recruited. The following SNPs in HNF1A gene were selected: rs1169286, rs1169288, rs7979478, and rs2259816. Through linear or logistic regression analysis, we analyzed their association with T1D susceptibility and related clinical traits, such as insulin dose-adjusted glycated hemoglobin A1c (IDAA1c) and glycated hemoglobin (HbA1c). We found that rs1169286 was associated with IDAA1c and HbA1c values (p-value = 0.0027 and p-value = 0.0075, respectively), while rs1169288 was associated with IDAA1c (p-value = 0.0081). No association between HNF1A SNPs and T1D development emerged. In conclusion, our findings suggest for the first time that HNF1A variants may be a risk factor for beta cell function and glycaemic control in T1D individuals.

1750-P: Differential Impacts of Two Novel HNF1A Variants Associated with Familial Young-Onset Diabetes on Protein Function and Insulin Secretion In Vitro

Introduction: Mutations in the HNF1A are a cause of monogenic diabetes and are usually dominantly inherited. We report in vitro and clinical studies on two novel HNF1A variants to determine pathogenicity: a recessive (homozygous) HNF1A variant (p.A251T) and a dominant HNF1A variant (p.S19L). We also investigated the mechanisms by which the former variant affects insulin secretion using beta cells derived from induced pluripotent stem cells (iPSCs). Methods: Nuclear localization, DNA binding, and transactivation activity were examined using standard techniques. iPSC lines from proband carrying the p.A251T variant were differentiated into beta-like cells according to Sui et al, CPHG, 2018. Ca2+ dynamics were monitored with Cal-520, and insulin secretion was measured by homogeneous time-resolved fluorescence assay compared to wild-type HNF1A (WT). Results: HNF1A p.A251T showed a mild (10.4% ± 7.3, p= 0.16) decrease in transactivation activity compared with WT, and decreased (48% of WT, p=0.12) DNA binding. These reductions were significant in p.S19L (DNA binding 57% of WT, p=0.049). Significant impaired nuclear uptake (63% vs 90% cells for WT, p<0.0001) was only apparent for p.S19L. Glucose-stimulated insulin secretion was decreased in A251T iPSCs compared with WT (0.29% vs. 0.46% of total insulin). In contrast, glucose-induced Ca2+ increases were increased in mutant cells (arbitrary units: 7.39 ± 1.23, mean +/− SD, n=725 cells vs. 4.58 ± 0.77, n=724, p= < 0.001) Conclusion: We demonstrate the value of combining multiple in vitro functional studies to decipher novel HNF1A variant pathogenicity. The recessive p.A251T variant has relatively mild effects on protein function compared to a dominant p.S19L variant. However, when studying proband iPSC derived beta-cell A251T impairs glucose-stimulated insulin secretion despite preserved Ca2+ entry, suggesting a novel mechanism of action not captured by standard tests of HNF1A function. Disclosure I.Cherkaoui: None. G.A.Rutter: Consultant; Sun Pharmaceutical Industries Ltd. Q.Du: None. C.Dion: None. H.Leitch: None. P.L.Chabosseau: None. D.M.Egli: None. D.Sachedina: None. J.Wastin: None. S.Misra: Research Support; Dexcom, Inc. Funding Diabetes UK

Hepatocellular Adenoma: Report of 2 Cases That Highlight the Relevance of Phenotype-Genotype Correlation in the Pediatric Population.

Hepatocellular adenoma (HCA) in the pediatric population is very rare and there are only limited studies, especially with molecular characterization of the tumors. Main HCA subtypes recognized in the current WHO classification include HNF1A-inactivated HCA (H-HCA), inflammatory HCA (IHCA), β-catenin-activated HCA (b-HCA), and β-catenin-activated IHCA (b-IHCA) and sonic hedgehog HCA (shHCA) is reported as an emerging subtype. Clinical history, pathological information, and molecular studies for a series of 2 cases of pediatric HCA were reviewed. Case 1 was a b-HCA characterized by somatic CTNNB1 S45 mutation in a 11-year-old male with Abernethy malformation. Case 2 was a H-HCA characterized by germline HNF1A variant (c.526+1G>A) in a 15-year-old male associated with maturity-onset diabetes of the young type 3 (MODY3). Our findings highlight the rarity of these 2 cases associated with adenomatosis, and the contribution of molecular/genetic analysis for proper sub-typing, prognosis and family surveillance.

Molecular characterization and re-interpretation of HNF1A variants identified in Indian MODY subjects towards precision medicine.

HNF1A is an essential component of the transcription factor network that controls pancreatic β-cell differentiation, maintenance, and glucose stimulated insulin secretion (GSIS). A continuum of protein malfunction is caused by variations in the HNF1A gene, from severe loss-of-function (LOF) variants that cause the highly penetrant Maturity Onset Diabetes of the Young (MODY) to milder LOF variants that are far less penetrant but impart a population-wide risk of type 2 diabetes that is up to five times higher. Before classifying and reporting the discovered variations as relevant in clinical diagnosis, a critical review is required. Functional investigations offer substantial support for classifying a variant as pathogenic, or otherwise as advised by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) ACMG/AMP criteria for variant interpretation. To determine the molecular basis for the variations in the HNF1A gene found in patients with monogenic diabetes in India. We performed functional protein analyses such as transactivation, protein expression, DNA binding, nuclear localization, and glucose stimulated insulin secretion (GSIS) assay, along with structural prediction analysis for 14 HNF1A variants found in 20 patients with monogenic diabetes. Of the 14 variants, 4 (28.6%) were interpreted as pathogenic, 6 (42.8%) as likely pathogenic, 3 (21.4%) as variants of uncertain significance, and 1 (7.14%) as benign. Patients harboring the pathogenic/likely pathogenic variants were able to successfully switch from insulin to sulfonylureas (SU) making these variants clinically actionable. Our findings are the first to show the need of using additive scores during molecular characterization for accurate pathogenicity evaluations of HNF1A variants in precision medicine.

Monogenic diabetes in New Zealand - An audit based revision of the monogenic diabetes genetic testing pathway in New Zealand.

To evaluate (a) the diagnostic yield of genetic testing for monogenic diabetes when using single gene and gene panel-based testing approaches in the New Zealand (NZ) population, (b) whether the MODY (Maturity Onset Diabetes of the Young) pre-test probability calculator can be used to guide referrals for testing in NZ, (c) the number of referrals for testing for Māori/Pacific ethnicities compared to NZ European, and (d) the volume of proband vs cascade tests being requested. A retrospective audit of 495 referrals, from NZ, for testing of monogenic diabetes genes was performed. Referrals sent to LabPlus (Auckland) laboratory for single gene testing or small multi-gene panel testing, or to the Exeter Genomics Laboratory, UK, for a large gene panel, received from January 2014 - December 2021 were included. Detection rates of single gene, small multi-gene and large gene panels (neonatal and non-neonatal), and cascade testing were analysed. Pre-test probability was calculated using the Exeter MODY probability calculator and ethnicity data was also collected. The diagnostic detection rate varied across genes, from 32% in GCK, to 2% in HNF4A, with single gene or small gene panel testing averaging a 12% detection rate. Detection rate by type of panel was 9% for small gene panel, 23% for non-neonatal monogenic diabetes large gene panel and 40% for neonatal monogenic diabetes large gene panel. 45% (67/147) of patients aged 1-35 years at diabetes diagnosis scored <20% on MODY pre-test probability, of whom 3 had class 4/5 variants in HNF1A, HNF4A or HNF1B. Ethnicity data of those selected for genetic testing correlated with population diabetes prevalence for Māori (15% vs 16%), but Pacific People appeared under-represented (8% vs 14%). Only 1 in 6 probands generated a cascade test. A new monogenic diabetes testing algorithm for NZ is proposed, which directs clinicians to choose a large gene panel in patients without syndromic features who score a pre-test MODY probability of above 20%.

Evidence for diet-gene interaction in early-onset colorectal cancer.

182 Background: About 10 percent of all colorectal cancers are in subjects who are not yet 50 (EO-CRC) and the occurrence of early onset colorectal cancer (EO-CRC) is rising in the US. Patients with CRC are twice as likely to have diabetes or be overweight. Using targeted exome sequencing of germline DNA from EO-CRC subjects, we identified a missense mutation at Ala98Val within the DNA binding domain of Hepatic Nuclear Factor 1 alpha (HNF1A, 12q24.31, Rs1800574). HNF1A is the most frequently mutated gene in diabetic individuals whose onset of diabetes occurs typically before age 25 (MODY3 locus). Our aim was to demonstrate that the HNF1A variant provides a genomic landscape for EO-CRC to develop in the setting of a specific diet type. Methods: HNF1A was identified using targeted exon sequencing of archived leukocyte DNA from subjects with EO-CRC. Flag-tagged WT and HNF1AA98V expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts were prepared for Electrophoretic Mobility Shift Assays (EMSAs) to test binding differences. An Hnf1aA98V mutant mouse model was generated using CRISPR/Cas9. WT, Hnf1a A98V/+ and Hnf1a A98V/A98V mice were placed on 3 diets--normal chow, a high fat (~34%) diet (HFD) or a high sugar (~62% fructose) diet (HSD) after weaning at 3 weeks. The mice were followed for 12 months, weighed monthly, and observed for clinical signs of morbidity. After euthanizing, blood, colon and liver were collected for histology and qPCR. Results: HNF1AA98V was identified in 13/145 subjects with EO-CRC. An additional 3 subjects exhibited mutations elsewhere in HNF1A. Flag-tagged WT and mutant HNF1A-expressing plasmids were transfected into HCT116 colon cancer cells and nuclear extracts showed reduced HNF1AA98V binding to its consensus DNA element by EMSA. Only heterozygous or homozygous Hnf1aA98V mice on the HFD showed a significant number of colon polyps (~19%). Only 1/22 heterozygous mice developed a polyp on the HSD. RNA-Seq analysis of colonic mucosa from the homozygous mutant compared to WT mice revealed an increase lipid and b-catenin regulated genes. Specifically, there was an increase in β-catenin, LEF 1 and MYC by immunohistochemistry in the polyp of HNF1AA98V, but not in the adjacent normal tissue. Liver steatosis in the Hnf1aA98V versus WT mice on the HFD was observed. Conclusions: Occurrence of the HNF1AA98V variant is increased among a cohort of EO-CRC patients and is a loss of function mutation that creates a genetic landscape for colon polyps on a HFD.

A novel splice-affecting HNF1A variant with large population impact on diabetes in Greenland.

The genetic disease architecture of Inuit includes a large number of common high-impact variants. Identification of such variants contributes to our understanding of the genetic aetiology of diseases and improves global equity in genomic personalised medicine. We aimed to identify and characterise novel variants in genes associated with Maturity Onset Diabetes of the Young (MODY) in the Greenlandic population. Using combined data from Greenlandic population cohorts of 4497 individuals, including 448 whole genome sequenced individuals, we screened 14 known MODY genes for previously identified and novel variants. We functionally characterised an identified novel variant and assessed its association with diabetes prevalence and cardiometabolic traits and population impact. We identified a novel variant in the known MODY gene HNF1A with an allele frequency of 1.9% in the Greenlandic Inuit and absent elsewhere. Functional assays indicate that it prevents normal splicing of the gene. Thevariant caused lower 30-min insulin (β=-232pmol/L, βSD=-0.695, P=4.43×10-4) and higher 30-min glucose (β=1.20mmol/L, βSD=0.441, P=0.0271) during an oral glucose tolerance test. Furthermore, the variant was associated with type 2 diabetes (OR 4.35, P=7.24×10-6) and HbA1c (β=0.113 HbA1c%, βSD=0.205, P=7.84×10-3). The variant explained 2.5% of diabetes variance in Greenland. The reported variant has the largest population impact of any previously reported variant within a MODY gene. Together with the recessive TBC1D4 variant, we show that close to 1 in 5 cases of diabetes (18%) in Greenland are associated with high-impact genetic variants compared to 1-3% in large populations. Novo Nordisk Foundation, Independent Research Fund Denmark, and Karen Elise Jensen's Foundation.