MODIS Research Articles

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Transcription Factors HNF1A, HNF4A, and FOXA2 Regulate Hepatic Cell Protein N-Glycosylation

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the-art clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9) molecular tool for the downregulation of the HNF1A, HNF4A, and FOXA2 genes in HepG2 cells—a human liver cancer cell line. The results show that the downregulation of all three genes individually and in pairs affects the transcriptional activity of many glyco-genes, although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures. The effect is better seen as an overall change in the total HepG2 N-glycome, primarily due to the extension of biantennary glycans. We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure. We also propose a model showing feedback loops with the mutual activation of HNF1A–FOXA2 and HNF4A–FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

Hepatotoxicity of cyanotoxin microcystin-LR in human: Insights into mechanisms of action in the 3D culture model Hepoid-HepaRG

Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

Premature coronary artery disease and severe hypertriglyceridemia in a patient with monogenic diabetes: a case report

Aim. Maturity- onset diabetes of the young (MODY) is a group of monogenic diseases with an autosomal dominant inheritance. This is a rarely diagnosed disease, occurring in 1 of 10 thousand adults and in 1 of 23 thousand children. Brief description. We present a case report of a patient with a monogenic diabetes HNF4A-MODY, severe hypertriglyceridemia, progressive premature coronary artery disease and a HNF4A gene pathogenic variant (ENST00000316099.8:c.1145dup).Discussion. This case demonstrates the effectiveness of triple hypoglycemic therapy, including empagliflozin for the treatment of monogenic diabetes. Despite the fact that the patient with diabetes reached the target glycated hemoglobin level, the combination of the disease with other cardiovascular risk factors (hypertension, hypertriglyceridemia, obesity, smoking, male sex) led to the progression of coronary and peripheral atherosclerosis. Considering the data of genetic testing, returning to sulfonylurea therapy should be considered. It is important for patient to follow a strict lipid-lowering diet and taking combination lipid-lowering therapy (rosuvastatin, fenofibrate and omega-3 polyunsaturated fatty acids) in order to achieve the target level of low-density lipoprotein cholesterol <1,4 mmol/l and triglycerides <1,7 mmol/l.

Genotype-phenotype correlation in Taiwanese children with diazoxide-unresponsive congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is a group of clinically and genetically heterogeneous disorders characterized by dysregulated insulin secretion. The aim of the study was to elucidate genetic etiologies of Taiwanese children with the most severe diazoxide-unresponsive CHI and analyze their genotype-phenotype correlations. We combined Sanger with whole exome sequencing (WES) to analyze CHI-related genes. The allele frequency of the most common variant was estimated by single-nucleotide polymorphism haplotype analysis. The functional effects of the ATP-sensitive potassium (KATP) channel variants were assessed using patch clamp recording and Western blot. Nine of 13 (69%) patients with ten different pathogenic variants (7 in ABCC8, 2 in KCNJ11 and 1 in GCK) were identified by the combined sequencing. The variant ABCC8 p.T1042QfsX75 identified in three probands was located in a specific haplotype. Functional study revealed the human SUR1 (hSUR1)-L366F KATP channels failed to respond to intracellular MgADP and diazoxide while hSUR1-R797Q and hSUR1-R1393C KATP channels were defective in trafficking. One patient had a de novo dominant mutation in the GCK gene (p.I211F), and WES revealed mosaicism of this variant from another patient. Pathogenic variants in KATP channels are the most common underlying cause of diazoxide-unresponsive CHI in the Taiwanese cohort. The p.T1042QfsX75 variant in the ABCC8 gene is highly suggestive of a founder effect. The I211F mutation in the GCK gene and three rare SUR1 variants associated with defective gating (p.L366F) or traffic (p.R797Q and p.R1393C) KATP channels are also associated with the diazoxide-unresponsive phenotype.

Differential spatial distribution of HNF4α isoforms during dysplastic progression of intraductal papillary mucinous neoplasms of the pancreas

Hepatocyte Nuclear Factor 4-alpha (HNF4α) comprises a nuclear receptor superfamily of ligand-dependent transcription factors that yields twelve isoforms in humans, classified into promoters P1 or P2-associated groups with specific functions. Alterations in HNF4α isoforms have been associated with tumorigenesis. However, the distribution of its isoforms during progression from dysplasia to malignancy has not been studied, nor has it yet been studied in intraductal papillary mucinous neoplasms, where both malignant and pre-malignant forms are routinely clinically identified. We examined the expression patterns of pan-promoter, P1-specific, and P2-specific isoform groups in normal pancreatic components and IPMNs. Pan-promoter, P1 and P2 nuclear expression were weakly positive in normal pancreatic components. Nuclear expression for all isoform groups was increased in low-grade IPMN, high-grade IPMN, and well-differentiated invasive adenocarcinoma. Poorly differentiated invasive components in IPMNs showed loss of all forms of HNF4α. Pan-promoter, and P1-specific HNF4α expression showed shifts in subnuclear and sub-anatomical distribution in IPMN, whereas P2 expression was consistently nuclear. Tumor cells with high-grade dysplasia at the basal interface with the stroma showed reduced expression of P1, while P2 was equally expressed in both components. Additional functional studies are warranted to further explore the mechanisms underlying the spatial and differential distribution of HNF4α isoforms in IPMNs.

Identification of causative gene variants for patients with known monogenic diabetes using a targeted next-generation sequencing panel in a single-center study.

We aimed to verify the usefulness of targeted next-generation sequencing (NGS) technology for diagnosing monogenic diabetes in a single center. We designed an amplicon-based NGS panel targeting 34 genes associated with known monogenic diabetes and performed resequencing in 56 patients with autoantibody-negative diabetes mellitus diagnosed at < 50years who had not been highly obese. By bioinformatic analysis, we filtered significant variants based on allele frequency (< 0.005 in East Asians) and functional prediction. We estimated the pathogenicity of each variant upon considering the family history. Overall, 16 candidate causative variants were identified in 16 patients. Among them, two previously known heterozygous nonsynonymous single-nucleotide variants associated with monogenic diabetes were confirmed as causative variants: one each in the GCK and WFS1 genes. The former was found in two independent diabetes-affected families. Two novel putatively deleterious heterozygous variants were also assumed to be causative from the family history: one frameshift and one nonsynonymous single-nucleotide variant in the HNF4A gene. Twelve variants remained as candidates associated with the development of diabetes. Targeted NGS panel testing was useful to diagnose various forms of monogenic diabetes in combination with familial analysis, but additional ingenuity would be needed for practice. The online version contains supplementary material available at 10.1007/s13340-023-00669-3.

Structural properties of the HNF-1A transactivation domain.

Hepatocyte nuclear factor 1α (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic β-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic β-cells.

Molecular diagnosis in patients with monogenic diabetes mellitus, and detection of a novel candidate gene

AimWe aimed to investigate molecular genetic basis of monogenic diabetes (DM) and novel responsible candidate genes with targeted Next Generation Sequencing (NGS) and Whole Exome Sequencing (WES). MethodsA hundred cases presenting with clinical findings and a family history of monogenic DM were included in the study. Molecular analysis was performed using an NGS panel including 14 genes. Following targeted NGS, WES was planned in cases in whom no variant was detected. ResultsThirty different disease-causing variants in seven different genes were detected in thirty-five (35 %) cases with targeted NGS approach. Most common pathogenic variant was found in GCK gene in 25 (25 %) cases. Four different variants were detected in 4 (4 %) patients in ABCC8 gene. In 45 of 65 cases; WES analyses were done. A heterozygous c.2635C > T(p.Gln879Ter) variant was detected in IFIH1 gene in a patient with incidental hyperglycemia. In the segregation analysis affected mother was shown to be heterozygous for the same variant. ConclusionMolecular etiology was determined in 35 % cases with the NGS targeted panel. Seventeen novel variants in monogenic DM genes have been identified. A candidate gene determined by WES analysis in a case that could not be diagnosed with NGS panel in this study.

Inhibiting HNF4A suppresses inflammation whilst promoting trophoblast invasion and migration: A promising target for the treatment of preeclampsia

Preeclampsia (PE) is a complex disease of pregnancy, and an important cause of this disease is insufficient trophoblast invasion and migration. However, the underlying mechanism of PE remains largely unknown. Here, transcriptome sequencing analysis found the high expression of hepatocyte nuclear factor 4 alpha (HNF4A) in PE placentas. Meanwhile, we found that HNF4A expression was up-regulated in the placentas of PE patients. Thus, we assumed that HNF4A might be involved in PE progression. To validate our hypothesis, l-arginine methyl ester (l-NAME) or lipopolysaccharide (LPS)-treated rats were used to mimic the pathological status of PE in vivo. Consistently, HTR8/SVneo cells were treated with hypoxia/reoxygenation (H/R) or LPS to simulate PE progression in vitro. The results observed an increase in elevated urine protein levels, systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), which indicated that the PE-like rat model was successfully established. Meanwhile, the expression of pro-inflammatory cytokines interleukin (IL)-6 and IL-1β was increased in PE placentas. HTR8/SVneo cells were used to further explore the underlying mechanism of PE in vitro. H/R conditions up-regulated the acetylation level of HNF4A. Further analysis showed that HNF4A overexpression inhibited trophoblast invasion and migration, while HNF4A knockdown promoted the progression. Additionally, inhibiting HNF4A was found to reduce the levels of IL-6 and IL-1β secretion in HTR8/SVneo cells following H/R or LPS exposure. Conclusively, these findings suggest that inhibiting HNF4A suppresses inflammation whilst promoting trophoblast invasion and migration in PE, providing a promising target for the treatment of PE.

FRI637 Missed MODY With Complex Mutation

Abstract Disclosure: S. Bulchandani: None. S. Majety: None. P. Kundra: None. Introduction: The prevalence range of Maturity-Onset Diabetes of the Young (MODY) is 1-5% among all patients diagnosed with diabetes. Strong family history of diabetes is suggestive of autosomal dominant inheritance. The symptoms of MODY range from mild asymptomatic hyperglycemia to overt insulinopenia requiring insulin therapy. Clinical Case: 33-year-old male presented to the endocrinology clinic for evaluation. He was diagnosed with type 2 diabetes mellitus at the age of 13 years and was initially treated with insulin which was subsequently transitioned to repaglinide and metformin. Reported history of hypoglycemia at birth in self and recently born daughter. Family History was significant for diabetes mellitus diagnosed in biological father at the age of 16 years. Genetic testing for monogenic diabetes revealed pathogenic variant in hepatocyte nuclear factor 4 alpha (HNF4a) and variant of unknown significance in Paired Box Gene 4 (PAX4). Therapy was switched to glipizide 2.5 mg orally which resulted in adequate glycemic control. Genetic testing was recommended for his daughter. Conclusion: MODY is a rare condition and because of its wide clinical presentation, it is common for it to be missed. There are about 14 subtypes of MODY with different genetic mutations, each affecting the beta cell function in a different way. Our case is unique given that our patient had two genetic mutations. Amongst all forms of MODY, HNF4a with a prevalence of 5% to 10% is the third most common mutation of MODY. PAX4 is a transcription factor that is critical for the formation of beta cells, and is important as a regulator in commitment of progenitor cells to mature islet cells. Mutations in PAX gene associated with monogenic diabetes were first identified in two patients of Thai origin, who did not present with mutations in the other known MODY genes. There should be high vigilance and low threshold for genetic testing for MODY in patients who have high likelihood of possessing a form of MODY, particularly as the treatment can be tailored to the clinical presentation of the patient.

SAT092 A Case Of Mature Onset Diabetes Of The Young

Abstract Disclosure: L. Beshay: None. A. Thakkar: None. Background: Maturity onset diabetes of the young (MODY) is a disorder characterized by noninsulin-dependent diabetes diagnosed at a young age (&amp;lt;25 years) with autosomal dominant inheritance and lack of autoantibodies. It accounts for 2 to 5 percent of diabetes cases. Several different genetic abnormalities have been identified causing disruption in pancreatic beta cells development and function leading to impaired insulin secretion. Mutations in hepatocyte nuclear factor-1-alpha (HNF1A) is the most commonly identified mutation occurring in 52 to 65 of MODY cases. Case Description: A 21 year old female with a history of diabetes and recurrent skin abscesses presented to the emergency room with right lower quadrant epidermal abscess. Endocrinology was consulted for evaluation of her diabetes. She was diagnosed at age 14 when she presented with weight loss, polyuria, polydipsia, BG 406 without diabetic ketoacidosis. She reported a history of type 2 diabetes in mother. Her medications included metformin, lantus and humalog. It was noted that her diabetes was never controlled with an A1c persistently &amp;gt;10%. Patient reported skin swelling with insulin injections and working two jobs which limited her ability to take more than one injection per day. Her BMI is 23 kg/m2. Labs this admission showed HbA1c 13.7%, GAD 65 Ab and IA-2 Ab negative, c-peptide 1.1 ng/ml, with a glucose of 209 mg/dl. MODY was suspected due to her age, intact c-peptide, and lack of DM1 antibodies. The patient was discharged on Trulicity 0.75 mg weekly, Lantus 12U nightly, Glipizide 2.5mg daily. On follow up, a MODY genetic panel confirmed a HNF1A missense variant with AD inheritance. Since initiation of sulfonylurea, she has had improved control of diabetes. Glipizide dose is being titrated up with concomitant decrease in insulin dose with plan to discontinue basal insulin once her blood sugars are at goal. Conclusion: The HNF1A gene on chromosome 12 is a weak transactivator of the insulin gene in beta cells. Mutations of HNF1A can lead to abnormal insulin secretion and a low renal threshold for glucose. These patients are at risk for micro- and macrovascular complications of diabetes. In terms of treatment, they exhibit increased insulin sensitivity and marked sensitivity to the hypoglycemic effects of sulfonylureas compared with patients with type 2 diabetes. Thus, patients can be successfully treated with sulfonylurea monotherapy, and in one clinical study, approximately 70 percent of patients previously treated with insulin successfully switched to sulfonylureas. Thus, it is important to evaluate etiology of diabetes, as detection of MODY variants in which sulfonylureas are used for treatment simplifies management and improves outcomes as demonstrated in this case. Presentation: Saturday, June 17, 2023

SAT165 A Novel Mutation In The HNF4α Gene (C.691C&amp;gt;T, P.arg231trp) Likely To Be Pathogenic For Maturity Onset Diabetes Of The Young Type 1 Presenting With Hyperinsulinemic Hypoglycemia In An Infant

Abstract Disclosure: A. Rodriguez: None. S. Sastry: None. W. Chemaitilly: None. N. Gurtunca: None. Background: Maturity Onset Diabetes of the Young type 1 (MODY 1) is a monogenic form of diabetes mellitus caused by a defective HNF4α gene, resulting in reduced insulin secretion and diabetes in early adolescence and young adulthood. MODY 1 is also known to be associated with increased insulin secretion during fetal, neonatal and childhood periods with consequences that range from fetal macrosomia to persistent hyperinsulinism. Traditionally, HNF4α gene mutations represents 5% of all cases of diazoxide-responsive hyperinsulinism. Clinical Case: A 1 month old male born full term and large for gestational age (birth weight of 4920g) following an uncomplicated pregnancy and delivery developed hypoglycemia soon after birth and required hospital admission on day of life 2 with a serum glucose of 30 mg/dL and an insulin level of 5 uIU/mL. A fasting challenge showed hypoglycemia at 9 hours with a serum glucose of 47 mg/dL and an insulin level of 10.3 uIU/mL confirming hyperinsulinemic hypoglycemia. He was started on diazoxide to which he responded well and was placed on a continuous glucose monitor (CGM). He was originally started on diazoxide 8mg/kg/day that later was decreased to 6.6mg/kg/day due to blood glucoses in the 200’s. He passed the fasting study 5 days after starting diazoxide and went home with the CGM with subsequent dose titration made with guidance from remote data sharing. A neonatal hypoglycemia panel was sent and showed a novel mutation, c.691C&amp;gt;T, p.ARG231Trp in the HNF4α gene classified as a variant of uncertain significance. Computer modeling software suggested that this gene might disrupts the gene structure and function. This variant is present in population databases at a 0.0009% frequency and this missense change has been observed in individuals with clinical features of maturity onset diabetes of the young. Patient’s father carries the same mutation and paternal grandmother carries a diagnosis of type 2 diabetes mellitus. Conclusion: This is a novel mutation that should be considered pathogenic based on this patient’s neonatal presentation and previous literature. This case also presents possible uses of CGM in infants with diazoxide responsive congenital hyperinsulinism in order to decrease burden from monitoring and streamline communication with the medical team Presentation: Saturday, June 17, 2023

Characterisation of HNF1A variants in paediatric diabetes in Norway using functional and clinical investigations to unmask phenotype and monogenic diabetes

Aims/hypothesisCorrectly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes.MethodsWe extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers’ phenotype and treatment response to sulfonylurea.ResultsIn total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic.Conclusions/interpretationBased on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY.Graphical

SAT078 A Case Of MODY Due To A Rare Activating Variant In The ABCC8 Gene

Abstract Disclosure: J. Milosavljevic: None. J.M. Greally: None. S. Milman: None. Background: ATP-binding cassette transporter subfamily C member 8 (ABCC8) gene encodes the sulfonylurea receptor 1 (SUR1) regulatory subunit of the beta cell ATP-sensitive K+ channel (KATP). Activating mutations in the ABCC8 cause both neonatal and maturity-onset diabetes of the young (MODY). The majority of patients (85%) with diabetes due to ABCC8 gene mutations could be successfully treated with oral sulfonylureas. A substitution c.2473C&amp;gt;T in the ABCC8 gene results in R825W change in the nucleotide-binding domain 1 of SUR1 and in activation of the KATP channel. We report a case of MODY with an activating mutation of ABCC8 gene (R825W). Clinical Case: A 23-year old woman with a history of ventricular septal defect was diagnosed with diabetes mellitus after she presented with a 2-month history of polyuria and polydipsia. At the time of diagnosis, random serum glucose level was 242 mg/dL, Hemoglobin A1c 7.6%, and BMI was 18 kg/m2. There was absence of acanthosis nigricans on exam or a family history of diabetes, although paternal medical history was unknown. A history of low birth weight was reported, but no developmental delays, neonatal diabetes or history of hypoglycemia. A history of seizure during childhood was also noted but was not associated with hypoglycemia. Additional work-up revealed absence of glutamic acid decarboxylase, islet cell, and insulin autoantibodies. C-peptide level was 2.0 ng/mL with blood glucose 113 mg/dL. The patient did not experience microvascular complications of diabetes. Genetic testing was negative for known mutations in HNF1A, HNF1B, HNF4A, GCK, and PPX1 genes. The patient was initially treated with metformin, but never achieved adequate glycemic control and experienced side effects. She was subsequently treated with sitagliptin and ertugliflozin, however, glycemic control remained inadequate (HbA1C 8.1%). At the age of 33 she was referred for clinical exome sequencing. This detected heterozygous ABCC8 variant c.2473 C&amp;gt;T; p.(R825W). Glucose control improved once therapy was switched to glipizide, resulting in the most recent HbA1C of 6.0%. Conclusion: Activating mutations of ABCC8 gene are infrequent genetic causes of diabetes in adults. Sequencing of the ABCC8 gene should be considered in select adult patients, as diagnosis can guide management decisions. Presentation: Saturday, June 17, 2023

Molecular cloning and tissue distribution of glucokinase and glucose-6-phosphatase catalytic subunit paralogs in largemouth bass Micropterus salmoides: Regulation by dietary starch levels and a glucose load

The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be one of the main reasons for the low glucose disposal of carnivorous fish, but is not yet well understood in largemouth bass Micropterus salmoides (LMB). In this study, the full length cDNA sequences of genes encoding glucokinase (Gck, catalyzing glucose phosphorylation) and glucose-6-phosphatase catalytic subunit (G6pc, catalyzing glucose dephosphorylation) were cloned by the RACE method from the liver of LMB. Subsequently, the distribution of g6pc and gck as well as their transcriptional regulation by dietary starch levels and a glucose load were investigated. Only one gck gene was identified, while the tandem duplication of g6pca.1 gene was named as g6pca.2 in LMB. The full cDNA sequences of g6pca.1, g6pca.2 and gck in LMB were 1585, 1813 and 2115 bp in length, encoding 478, 352 and 359 amino acids, respectively. Gck was predicted to contain two hexokinase domains, an ATP-binding domain and multiple functional sites, while G6pca.1 and G6pca.2 contained nine transmembrane helices, a PAP2 (type-2 phosphatidic acid phosphatase) domain and multiple functional amino acid sites. Both g6pca.1 and g6pca.2 were predominantly distributed in the liver and to some extent in the intraperitoneal fat, intestine and pyloric caeca, while gck was mainly transcribed in the liver and to some extent in the heart, intestine and brain. Both feeding a high starch diet and a glucose load stimulated the mRNA expression of gck in the liver of LMB. An increase of dietary starch from 9% to 14% down-regulated the transcription of g6pca.1 in the liver of LMB. However, both the mRNA levels of hepatic g6pca.1 and g6pca.2 were sharply up-regulated in LMB during 1–3 h after a glucose load. Overall, the results of this study suggested that the functions of G6pc (G6pca.1 and G6pca.2) and Gck in LMB were highly conserved in evolution. Though hepatic glucose-G6P interconversion was well regulated at the transcript level in LMB fed high starch diets, a futile cycle between glucose and G6P was induced in the liver after a glucose load.

The Effects of Retinoic Acid on the Expression of HNF4α and Cdx-2 Genes in Caco-2 Cells as a Model of Intestinal Barrier

The Effects of Retinoic Acid on the Expression of HNF4α and Cdx-2 Genes in Caco-2 Cells as a Model of Intestinal Barrier

An Italian MODY family with proband and son carrying variants in GCK and HFN1A: is it a true case of digenic MODY?

Maturity Onset Diabetes of the Young (MODY) is a monogenic autosomal dominant disorder affecting 1-5 % of all patients with diabetes mellitus. In Caucasians, GCK and HNF1A mutations are the most common cause of MODY. Here, we report two family members carrying a genetic variant of both GCK and HNF1A gene and their nine year clinical follow-up. Our report urges physicians to be cautious when variants in two genes are found in a single patient and suggests that collaboration with MODY genetics experts is necessary for correct diagnosis and treatment.

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.

Identification and precision therapy for three maturity-onset diabetes of the young (MODY) families caused by mutations in the HNF4A gene.

Heterozygous pathogenic variants in HNF4A gene cause maturity-onset diabetes of the young type 1 (MODY1). The mutation carriers for MODY1 have been reported to be relatively rare, in contrast to the most frequently reported forms of MODY2 and MODY3. Whole exome sequencing (WES) and Sanger sequencing were performed for genetic analysis of MODY pedigrees. Tertiary structures of the mutated proteins were predicted using PyMOL software. Three heterozygous missense mutations in the HNF4A gene, I159T, W179C, and D260N, were identified in the probands of three unrelated MODY families using WES, one of which (W179C) was novel. Cascade genetic screening revealed that the mutations co-segregated with hyperglycemic phenotypes in their families. The molecular diagnosis of MODY1 has partly transformed its management in clinical practice and improved glycemic control. The proband in family A successfully converted to sulfonylureas and achieved good glycemic control. Proband B responded well to metformin combined with diet therapy because of his higher body mass index (BMI). The proband in family C, with paternal-derived mutations, had markedly defective pancreatic β-cell function due to the superposition effect of T2DM susceptibility genes from the maternal grandfather, and he is currently treated with insulin. In silico analysis using PyMOL showed that the I159T and D260N mutations altered polar interactions with the surrounding residues, and W179C resulted in a smaller side chain. We identified three heterozygous missense mutations of HNF4A from Chinese MODY families. Structural alterations in these mutations may lead to defects in protein function, further contributing to the hyperglycemic phenotype of mutation carriers.