MODIS Research Articles

Molecular and clinical profiles of pediatric monogenic diabetes subtypes: comprehensive genetic analysis of 138 patients.

Single gene variants that give rise to neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY) and syndromic forms of diabetes mellitus (SDM) are responsible for 3.1-4.2% of all diabetes cases. This single-center study with a relatively larger sample size aimed to evaluate the clinical and genetic characteristics of Chinese children with suspected monogenic diabetes (MD) using next generation sequencing (NGS) methods. Data were collected from 1550 consecutive children diagnosed with diabetes/hyperglycemia at the Endocrinology Department of Children's Hospital of Nanjing Medical University from 2012 to 2023. The genotype and phenotype of 138 children with suspected MD were retrospectively analyzed. Among 138 children, 16, 97, and 25 patients with NDM, suspected MODY and SDM were assessed by NGS, with a pick-up rate of 87.5%, 57.8%, and 56%, respectively. In total, there was a high pick-up rate of MD, with 58% (80 of 138) among antibody-negative pediatric patients. Pathogenic variants were found in GCK, HNF1A, INS, KCNJ11, INSR, HNF4A, ABCC8, WFS1, ALMS1, HNF1B, BLK and ZFP57 genes with 13 novel variants in addition to 4 patients with CNVs. In this cohort, GCK-MODY was the leading cause and the mildest type of MODY. GCK-MODY displayed favorable lipid profile when compared to non-GCK-MODY and MODYX, which might be cardioprotective. Following an accurate genetic diagnosis of diabetes, 19 patients switched from insulin therapy to oral agents or lifestyle interventions. NGS tests helped to identify the precise etiology of monogenic diabetic patients which has implications for better individualized management.

8616 A Novel Mutation of Maturity Onset Diabetes of the Young

Abstract Disclosure: K.H. Weerasinghe: None. N. Branis: None. Background: Maturity onset diabetes of the young (MODY) is an autosomal dominant condition characterized by various subsets (Nkonge et al., 2020). A mutation in the glucokinase gene (GCK), otherwise known as MODY type 2, is often characterized by mild hyperglycemia managed with lifestyle modifications (Nkonge et al., 2020). Novel mutations in this gene may differ in their clinical course and need close clinical monitoring. Clinical Case: A 19 year old male presented for evaluation of his diabetes mellitus. In 2020, the patient was hyperglycemic on routine blood work, with asymptomatic features. His condition was managed with lifestyle modifications. The HbA1c was 6.8 and 7; while fructosamine was 338 mmol/L (normal 205-285 mmol/L). Subsequent workup revealed GAD 65 to be less than 5 U/ml(normal range being 0-5 U/ml) with negative islet antibodies. C-peptide was similarly measured and found to be 1.5 ng/ml, within the limits of 1.1-4.4 ng/ml, demonstrating a good pancreatic reserve. A MODY genetic profile was then conducted which revealed a novel mutation in the GCK gene, variant c.1064T>C (p.L355P). As this is a novel mutation, the probability of MODY was calculated, and found to be 75% using the Exeter maturity-onset diabetes of the young (MODY) probability calculator (EMPC).The patient’s clinical course thus far has been managed conservatively as his hyperglycemia has been mild. However, recently the patient’s post prandial sugars were found to be persistently elevated. Pharmacotherapy such as sulfonylureas was discussed however, as per the patient’s wishes, stricter dietary modifications will be applied prior to the initiation of medication. Conclusion: Patients with novel mutations should be closely monitored as the general expected clinical course may vary. These changes are demonstrated in this case report as the patient started developing elevation in postprandial sugar levels uncharacteristically. Close monitoring of the patient’s future clinical status will be essential in learning more about the differences in prognosis and long term management.

Protective effects of cilostazol via the HNF1α/FXR signalling pathway and anti-apoptotic mechanisms in a rat model of estrogen-induced intrahepatic cholestasis

Currently, there is a lack of targeted medications for estrogen-induced intrahepatic cholestasis (EIC) and the primary objective in managing this condition is to safeguard liver function. Consequently, this study was conducted to examine the pharmacological efficacy of cilostazol (CTZ) in the management of EIC and explore its underlying mechanisms through the use of an animal model. Thirty female Sprague-Dawley rats were divided into five groups of six animals each: Normal group, 17-ethinylestradiol (EE)-induced intrahepatic cholestasis group, EE + ursodeoxycholic acid (UDCA)-treated group, EE + CTZ (5 mg/kg)-treated group, and EE + CTZ (10 mg/kg)-treated group. It was found that the therapeutic efficacy of UDCA and low dosage of CTZ (5 mg/kg) was comparable. Nevertheless, when CTZ was administered at a dose of 10 mg/kg, it resulted in the normalization of all liver function parameters, oxidative stress, and pro-inflammatory markers, together with improvement in the histopathological derangements and hepatocytic apoptosis. These effects were mediated through the activation of the hepatocyte nuclear factor-1 alpha (HNF1α)/Farnesoid X receptor (FXR) pathway with subsequent down-regulation of the bile acids (BAs) synthesis enzyme; cholesterol 7α-hydroxylase (CYP7A1), and up-regulation of the BAs-metabolizing enzyme; cytochrome P450 (CYP)3A1 and the bile salt export pump; BSEP. Therefore, the administration of CTZ in a dose-dependent manner can protect against EIC through regulating the HNF1α/FXR pathway and anti-apoptotic mechanisms. This implies that CTZ exhibits considerable promise as a therapeutic agent for the treatment of cholestatic liver disorders.

Monogenic Defects of Beta Cell Function: From Clinical Suspicion to Genetic Diagnosis and Management of Rare Types of Diabetes

Monogenic defects of beta cell function refer to a group of rare disorders that are characterized by early-onset diabetes mellitus due to a single gene mutation affecting insulin secretion. It accounts for up to 5% of all pediatric diabetes cases and includes transient or permanent neonatal diabetes, maturity-onset diabetes of the young (MODY), and various syndromes associated with diabetes. Causative mutations have been identified in genes regulating the development or function of the pancreatic beta cells responsible for normal insulin production and/or release. To date, more than 40 monogenic diabetes subtypes have been described, with those caused by mutations in HNF1A and GCK genes being the most prevalent. Despite being caused by a single gene mutation, each type of monogenic diabetes, especially MODY, can appear with various clinical phenotypes, even among members of the same family. This clinical heterogeneity, its rarity, and the fact that it shares some features with more common types of diabetes, can make the clinical diagnosis of monogenic diabetes rather challenging. Indeed, several cases of MODY or syndromic diabetes are accurately diagnosed in adulthood, after having been mislabeled as type 1 or type 2 diabetes. The recent widespread use of more reliable sequencing techniques has improved monogenic diabetes diagnosis, which is important to guide appropriate treatment and genetic counselling. The current review aims to summarize the latest knowledge on the clinical presentation, genetic confirmation, and therapeutic approach of the various forms of monogenic defects of beta cell function, using three imaginary clinical scenarios and highlighting clinical and laboratory features that can guide the clinician in reaching the correct diagnosis.

Metastasis of colon cancer requires Dickkopf-2 to generate cancer cells with Paneth cell properties.

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2 -knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9 , a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

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.

Fibrotic liver extracellular matrix induces cancerous phenotype in biomimetic micro-tissues of hepatocellular carcinoma model

BackgroundDespite considerable advancements in identifying factors contributing to the development of hepatocellular carcinoma (HCC), the pathogenesis of HCC remains unclear. In many cases, HCC is a consequence of prolonged liver fibrosis, resulting in the formation of an intricate premalignant microenvironment. The accumulation of extracellular matrix (ECM) is a hallmark of premalignant microenvironment. Given the critical role of different matrix components in regulating cell phenotype and function, this study aimed to elucidate the interplay between the fibrotic matrix and malignant features in HCC. MethodsLiver tissues from both control (normal) and carbon tetrachloride (CCl4)-induced fibrotic rats were decellularized using sodium dodecyl sulfate (SDS) and Triton X-100. The resulting hydrogel from decellularized ECM was processed into micro-particles via the water-in-oil emulsion method. Micro-particles were subsequently incorporated into three-dimensional liver biomimetic micro-tissues (MTs) comprising Huh-7 cells, human umbilical vein endothelial cells (HUVECs), and LX-2 cells. The MTs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at day 11, immunofluorescence staining, immunoblotting, and spheroid migration assay at day 14 after co-culture. ResultsFibrotic matrix from CCl4-treated rat livers significantly enhanced the growth rate of the MTs and their expression of CCND1 as compared to the normal one. Fibrotic matrix, also induced the expression of epithelial-to-mesenchymal transition (EMT)-associated genes such as TWIST1, ACTA2, MMP9, CDH2, and VIMENTIN in the MTs as compared to the normal matrix. Conversely, the expression of CDH1 and hepatic maturation genes HNF4A, ALB, CYP3A4 was decreased in the MTs when the fibrotic matrix was used. Furthermore, the fibrotic matrix increased the migration of the MTs and their secretion of alpha-fetoprotein. ConclusionsOur findings suggest a regulatory role for the fibrotic matrix in promoting cancerous phenotype, which could potentially accelerate the progression of malignancy in the liver.

Correlations of Long Noncoding RNA HNF4A-AS1 Alternative Transcripts with Liver Diseases and Drug Metabolism.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. SIGNIFICANCE STATEMENT: This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations.

Re-diagnosis of diabetes mellitus type 1 to maturity-onset diabetes of the young type 2 (MODY 2) in a 26-year-old male: a case report

Diabetes mellitus (DM) encompasses a spectrum of metabolic disorders characterized by hyperglycemia due to defects in insulin secretion, action, or both. Type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are the most prevalent forms, whereas Maturity-Onset Diabetes of the Young (MODY) represents a monogenic subtype, often misdiagnosed due to its phenotypic overlap. This case study highlights a 26-year-old male initially diagnosed with T1DM at age 12, managed with insulin therapy. Upon admission, despite stable glycemic control and preserved C-peptide secretion, genetic testing revealed MODY 2 due to a variant in the GCK gene. Unlike T1DM and T2DM, MODY 2 is characterized by mild, persistent hyperglycemia with a low risk of complications, emphasizing the importance of genetic diagnosis for tailored management and family counseling. This case underscores the critical role of genetic evaluation in accurately diagnosing and managing atypical diabetes presentations.

MODY calculator applied in patients with clinical diagnosis of type 1 diabetes mellitus: Is a higher cutoff needed?

AimThis study aimed to evaluate the mean post-test probability (PTP) of the Maturity-onset diabetes of the young (MODY) calculator in a multiethnic cohort of patients previously diagnosed with type 1 diabetes (T1DM). Materials and methodsThe MODY probability calculator proposed by Shields and colleagues (2012) was applied to 117 patients from a T1DM outpatient clinic at a tertiary hospital in Brazil. Additionally, two exons of the HNF1A gene were sequenced in eight patients who hadn't received insulin treatment within six months after the diagnosis. Results17.1 % of patients achieved PTP >10 %; 11.1 % achieved PTP >25 % (and all patients >30 %), and 7.7 % achieved PTP >40 %. Among the patients who were selected for genetic sequencing, 100 % presented PTP >30 %, with 66.6 % achieving PTP >40 % and 41.6 % achieving PTP >75 %. These cutoffs are as suggested for the Brazilian population, according to previous investigations. No mutation was observed in the sequenced exons. ConclusionConsidering that only around 10 % of the evaluated cases achieved PTP >30 %, it is highly probable that the most suitable cutoff to select patients for genetic sequencing in a Brazilian cohort of T1DM is higher than the cutoff used in Caucasian populations.

Coinheritance of HNF1A and glucokinase variants in maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a group of monogenic forms of diabetes mellitus characterized by early-onset diabetes with dominant inheritance of beta-cell dysfunction. There are few reports of the coinheritance of glucokinase (GCK) and hepatocyte nuclear factor 1 alpha gene (HNF1A) variants underlying MODY in patients. Herein, we describe a case involving combinations of monoallelic GCK and HNF1A variants associated with MODY. A 10-year-old Japanese girl with a three-generation family history of diabetes without obesity showed high levels of urinary glucose during a school screening test. Her glucose metabolism profile revealed 124 mg/dL of fasting glucose, 6.9% glycated hemoglobin (HbA1c), and 2.78 ng/mL of C-peptide immunoreactivity levels. In a 75-g oral glucose tolerance test, her base glucose, peak glucose, insulin resistance, and homeostasis model assessment of beta cell function levels were 124 mg/dL, 210 mg/dL (120 min), 1.71, and 33%, respectively. Based on the clinical phenotype of GCK-MODY, alimentary and exercise therapy without oral hypoglycemic agents were used to maintain her fasting glucose and HbA1c levels. We explored the coinheritance of MODY with GCK and HNF1A variants in this and past cases and found that careful clinical follow-up is required to firmly establish phenotypic features. Moreover, the accumulation of data on genetically confirmed MODY associated with the coinheritance of GCK and HNF1A variants will be useful for understanding genotype-phenotype correlations. MODY is a group of monogenic forms of diabetes mellitus characterized by early-onset diabetes with the dominant inheritance of beta-cell dysfunction. MODY2 and MODY3 caused by heterozygous loss-of-function variants in the glucokinase (GCK) and hepatocyte nuclear factor 1 alpha (HNF1A) genes, respectively, are the most common forms of the disease. Few cases of MODY have previously been reported as being associated with the coinheritance of GCK and HNF1A variants. Careful clinical follow-up is required to firmly establish phenotypic features in the coinheritance of MODY with GCK and HNF1A variants. The accumulation of data on genetically confirmed MODY associated with the coinheritance of GCK and HNF1A variants will be useful for understanding genotype-phenotype correlations.

Molecular mechanism of HNF-1A-mediated HNF4A gene regulation and promoter-driven HNF4A-MODY diabetes.

Monogenic diabetes is a gateway to precision medicine through molecular mechanistic insight. Hepatocyte nuclear factor 1A (HNF-1A) and HNF-4A are transcription factors that engage in crossregulatory gene transcription networks to maintain glucose-stimulated insulin secretion in pancreatic β cells. Variants in the HNF1A and HNF4A genes are associated with maturity-onset diabetes of the young (MODY). Here, we explored 4 variants in the P2-HNF4A promoter region: 3 in the HNF-1A binding site and 1 close to the site, which were identified in 63 individuals from 21 families of different MODY disease registries across Europe. Our goal was to study the disease causality for these variants and to investigate diabetes mechanisms on the molecular level. We solved a crystal structure of HNF-1A bound to the P2-HNF4A promoter and established a set of techniques to probe HNF-1A binding and transcriptional activity toward different promoter variants. We used isothermal titration calorimetry, biolayer interferometry, x-ray crystallography, and transactivation assays, which revealed changes in HNF-1A binding or transcriptional activities for all 4 P2-HNF4A variants. Our results suggest distinct disease mechanisms of the promoter variants, which can be correlated with clinical phenotype, such as age of diagnosis of diabetes, and be important tools for clinical utility in precision medicine.

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.

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

Modulation of Glucose Consumption and Uptake in HepG2 Cells by Aqueous Extracts from the Coelomic Fluid of the Edible Holothuria tubulosa Sea Cucumber.

The cell-free aqueous extract from the coelomic fluid of Holothuria tubulosa was prepared and examined for its glucose-lowering effect on HepG2 cells in vitro. In particular, employing a combination of cytochemical, flow cytometric, PCR, and protein blot techniques, we evaluated its role on glucose internalization and storage and on the upregulation and surface translocation of the two glucose transporters GLUT-2 and -4. The changes in expression, synthesis, and/or activation of the GLUT2-related transcription factor hepatocyte nuclear factor-1 alpha (HNF1α) and the GLUT-4-translocation regulatory factors insulin receptor substrate-1 (IRS-1) and AKT were also studied. Our results showed the improved glucose response by HepG2 cells, leading to an evident increase in glucose consumption/uptake and glycogen storage upon exposure. Moreover, the extract induced molecular reprogramming involving the upregulation of (i) IRS1 gene expression, (ii) the transcription and translation levels of HNF1α, AKT, and GLUT-4, (iii) the phosphorylation level of AKT, (iv) the synthesis of GLUT-2 protein, and (v) the translocation of GLUT-2 and -4 transporters onto the plasma membrane. Cumulatively, our results suggest that the coelomic fluid extract from H. tubulosa can be taken into consideration for the development of novel treatment agents against diabetes mellitus.

Sex-Dimorphic Effects of Hypoglycemia on Metabolic Sensor mRNA Expression in Ventromedial Hypothalamic Nucleus-Dorsomedial Division (VMNdm) Growth Hormone-Releasing Hormone Neurons.

Growth hormone-releasing hormone (Ghrh) neurons in the dorsomedial ventromedial hypothalamic nucleus (VMNdm) express the metabolic transcription factor steroidogenic factor-1 and hypoglycemia-sensitive neurochemicals of diverse chemical structures, transmission modes, and temporal signaling profiles. Ghrh imposes neuromodulatory control of coexpressed transmitters. Multiple metabolic sensory mechanisms are employed in the brain, including screening of the critical nutrient glucose or the energy currency ATP. Here, combinatory laser-catapult-microdissection/single-cell multiplex qPCR tools were used to investigate whether these neurons possess molecular machinery for monitoring cellular metabolic status and if these biomarkers exhibit sex-specific sensitivity to insulin-induced hypoglycemia. Data show that hypoglycemia up- (male) or downregulated (female) Ghrh neuron glucokinase (Gck) mRNA; Ghrh gene silencing decreased baseline and hypoglycemic patterns of Gck gene expression in each sex. Ghrh neuron glucokinase regulatory protein (Gckr) transcript levels were respectively diminished or augmented in hypoglycemic male vs female rats; this mRNA profile was decreased by Ghrh siRNA in both sexes. Gene transcripts encoding catalytic alpha subunits of the energy monitor 5-AMP-activated protein kinase (AMPK), i.e., Prkaa1 and 2, were increased by hypoglycemia in males, yet only the former mRNA was hypoglycemia-sensitive in females. Ghrh siRNA downregulated baseline and hypoglycemia-associated Prkaa subunit mRNAs in males but elicited divergent changes in Prkaa2 transcripts in eu- vs hypoglycemic females. Results provide unique evidence that VMNdm Ghrh neurons express the characterized metabolic sensor biomarkers glucokinase and AMPK and that the corresponding gene profiles exhibit distinctive sex-dimorphic transcriptional responses to hypoglycemia. Data further document Ghrh neuromodulation of baseline and hypoglycemic transcription patterns of these metabolic gene profiles.

Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response.

The hepatic acute-phase response is characterized by a massive upregulation of serum proteins, such as haptoglobin and serum amyloid A, at the expense of liver homeostatic functions. Although the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has a well-established role in safeguarding liver function and its cistrome spans around 50% of liver-specific genes, its role in the acute-phase response has received little attention so far. We demonstrate that HNF4A binds to and represses acute-phase genes under basal conditions. The reprogramming of hepatic transcription during inflammation necessitates loss of HNF4A function to allow expression of acute-phase genes while liver homeostatic genes are repressed. In a pre-clinical liver organoid model overexpression of HNF4A maintained liver functionality in spite of inflammation-induced cell damage. Conversely, HNF4A overexpression potently impaired the acute-phase response by retaining chromatin at regulatory regions of acute-phase genes inaccessible to transcription. Taken together, our data extend the understanding of dual HNF4A action as transcriptional activator and repressor, establishing HNF4A as gatekeeper for the hepatic acute-phase response.

Enhancing fetal outcomes in GCK-MODY pregnancies: a precision medicine approach via non-invasive prenatal GCK mutation detection.

Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus. A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma. Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these. Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Identification and Functional Characterization of Alternative Transcripts of LncRNA HNF1A-AS1 and Their Impacts on Cell Growth, Differentiation, Liver Diseases, and in Response to Drug Induction.

The long non-coding RNA (lncRNA) hepatocyte nuclear factor-1 alpha (HNF1A) antisense RNA 1 (HNF1A-AS1) is an important lncRNA for liver growth, development, cell differentiation, and drug metabolism. Like many lncRNAs, HNF1A-AS1 has multiple annotated alternative transcripts in the human genome. Several fundamental biological questions are still not solved: (1) How many transcripts really exist in biological samples, such as liver samples and liver cell lines? (2) What are the expression patterns of different alternative HNF1A-AS1 transcripts at different conditions, including during cell growth and development, after exposure to xenobiotics (such as drugs), and in disease conditions, such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD) cirrhosis, and obesity? (3) Does the siRNA used in previous studies knock down one or multiple transcripts? (4) Do different transcripts have the same or different functions for gene regulation? The presented data confirm the existence of several annotated HNF1A-AS1 transcripts in liver samples and cell lines, but also identify some new transcripts, which are not annotated in the Ensembl genome database. Expression patterns of the identified HNF1A-AS1 transcripts are highly correlated with the cell differentiation of matured hepatocyte-like cells from human embryonic stem cells (hESC), growth and differentiation of HepaRG cells, in response to rifampicin induction, and in various liver disease conditions. The expression levels of the HNF1A-AS1 transcripts are also highly correlated to the expression of cytochrome P450 enzymes, such as CYP3A4, during HepaRG growth, differentiation, and in response to rifampicin induction.

Genetic diagnosis of endocrine disorders in Cyprus through the Cyprus Institute of Neurology and Genetics: an ENDO-ERN Reference Center

The report covers the current and past activities of the department Molecular Genetics-Function and Therapy (MGFT) at the Cyprus Institute of Neurology and Genetics (CING), an affiliated Reference Center for the European Reference Network on Rare Endocrine Conditions (Endo-ERN).The presented data is the outcome of > 15 years long standing collaboration between MGFT and endocrine specialists from the local government hospitals and the private sector. Up-to-date > 2000 genetic tests have been performed for the diagnosis of inherited rare endocrine disorders. The major clinical entities included Congenital Adrenal Hyperplasia (CAH) due to pathogenic variants in CYP21A2 gene and Multiple Endocrine Neoplasia (MEN) type 2 due to pathogenic variants in the RET proto-oncogene. Other rare and novel pathogenic variants in ANOS1, WDR11, FGFR1, RNF216, and CHD7 genes were also found in patients with Congenital Hypogonadotropic Hypogonadism. Interestingly, a few patients with Disorders of Sexual Differentiation (DSD) shared rare pathogenic variants in the SRD5A2, HSD17B3 and HSD3B2 while patients with Glucose and Insulin Homeostasis carried theirs in GCK and HNF1A genes. Lastly, MGFT over the last few years has established an esteemed diagnostic and research program on premature puberty with emphasis on the implication of MKRN3 gene on the onset of the disease and the identification of other prognosis biomarkers.As an Endo-ERN member MGFT department belongs to this large European network and holds the same humanistic ideals which aim toward the improvements of health care for patients with rare endocrine conditions in respect to improved and faster diagnosis.