Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Objective: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. Design: The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. Methods: We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Results: Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. Conclusion: These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

GCKIII (Germinal Center Kinase III) Kinases STK24 and STK25 (Serine/Threonine Kinase 24 and 25) Inhibit Cavernoma Development.

BACKGROUND: Cavernous cerebral malformations can arise because of mutations in the CCM1, CCM2, or CCM3 genes, and lack of Cdc42 has also been reported to induce these malformations in mice. However, the role of the CCM3 (cerebral cavernous malformation 3)-associated kinases in cavernoma development is not known, and we, therefore, have investigated their role in the process. METHODS: We used a combination of an in vivo approach, using mice genetically modified to be deficient in the CCM3-associated kinases STK24 and STK25 (serine/threonine kinases 24 and 25), and the in vitro model of human endothelial cells in which expression of STK24 and STK25 was inhibited by RNA interference. RESULTS: Mice deficient for both Stk24 and Stk25, but not for either of them individually, developed aggressive vascular lesions with the characteristics of cavernomas at an early age. Stk25 deficiency also gave rise to vascular anomalies in the context of Stk24 heterozygosity. Human endothelial cells deficient for both kinases phenocopied several of the consequences of CCM3 loss, and single STK25 deficiency also induced KLF2 expression, Golgi dispersion, altered distribution of ß-catenin, and appearance of stress fibers. CONCLUSIONS: The CCM3-associated kinases STK24 and STK25 play a major role in the inhibition of cavernoma development.

Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma.

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Recognition of maturity-onset diabetes of the young in China.

AIMS/INTRODUCTION: Given that mutations related to maturity-onset diabetes of the young (MODY) are rarely found in Chinese populations, we aim to characterize the mutation spectrum of MODY pedigrees. MATERIALS AND METHODS: Maturity-onset diabetes of the young candidate gene- or exome-targeted capture sequencing was carried out in 76 probands from unrelated families fulfilling the clinical diagnostic criteria for MODY. MAF <0.01 in the GnomAD or ExAC database was used to filter significant variants. Sanger sequencing was then carried out to validate findings. Function prediction by SIFT, PolyPhen-2 and PROVEAN or CADD was carried out in missense mutations. RESULTS: A total of 32 mutations in six genes were identified in 31 families, accounting for 40.79% of the potential MODY families. The MODY subtype detection rate was 18.42% for GCK, 15.79% for HNF1A, 2.63% for HNF4A, and 1.32% for KLF11, PAX4 and NEUROG3. Seven nonsense/frameshift mutations and four missense mutations with damaging prediction were newly identified novel mutations. The clinical features of MODY2, MODY3/1 and MODYX are similar to previous reports. Clinical phenotype of NEUROG3 p.Arg55Glufs*23 is characterized by hyperglycemia and mild intermittent abdominal pain. CONCLUSIONS: This study adds to the emerging pattern of MODY epidemiology that the proportion of MODY explained by known pathogenic genes is higher than that previously reported, and found NEUROG3 as a new causative gene for MODY.

Long-Term Observation of a Man With Severe Obesity and Undiagnosed Monogenic Diabetes Serendipitously Treated With Metabolic Surgery.

A 43-year-old man, with severe obesity (43 kg/m2) and diabetes (presumed as type 2 diabetes [T2D]), underwent vertical sleeve gastrectomy in 2009 and Roux-en-Y gastric bypass in 2013. Recently, whole exome sequencing (conducted to search for monogenic obesity) serendipitously revealed that the individual harbored a heterozygous glucokinase (GCK) variant p.(Arg422Leu) that was bioinformatically strongly predicted to be likely pathogenic. Therefore, he is likely to have concomitant maturity-onset diabetes of the young (MODY) type 2 (GCK-MODY). A retrospective evaluation of the clinical data showed that the subject was diagnosed with T2D (given his severe obesity) in 2005 and was treated with oral antidiabetic monotherapy. His hyperglycemia was mostly mild (HbA1c [hemoglobin] < 8.1%), consistent with that of MODY2, despite severe obesity. After vertical sleeve gastrectomy, complete diabetes remission (HbA1c <6.0% and fasting plasma glucose <5.6 mmol/L without use of antidiabetic medication) was achieved. The percentage of maximum body weight loss attained after surgery was 23.6%. Euglycemia was maintained during the subsequent decade, up to the last follow-up in 2019, without any sign of hypoglycemia. In conclusion, we report a decade-long clinical experience of a man with severe obesity and diabetes likely due to the coexistence of GCK-MODY and T2D, serendipitously treated with metabolic surgery. Interestingly, metabolic surgery was effective and safe for him.

Rab8 and Rabin8-Mediated Tumor Formation by Hyperactivated EGFR Signaling via FGFR Signaling.

The epidermal growth factor receptor (EGFR) signaling is important for normal development, such as vulval development in Caenorhabditis elegans, and hyperactivation of the EGFR is often associated with cancer development. Our previous report demonstrated the multivulva (Muv) phenotype, a tumor model in C. elegans (jgIs25 strain) by engineering LET-23/EGFR with a TKI-resistant human EGFR T790-L858 mutant. Because Rab proteins regulate vesicle transport, which is important for receptor signaling, we screened the RNAi in the jgIs25 strain to find the Rabs critical for Muv formation. Herein, we show that rab-8 RNAi and the rab-8 (-/-) mutation effectively reduce Muv formation. We demonstrate that RABN-8, an ortholog of Rabin8, known as a GEF for Rab8, is also required for Muv formation by promoting the secretion of EGL-17/FGF from vulval precursor cells. In addition, FGFR inhibitors decreased Muv formation mediated by mutant EGFR. Our data suggest that Rab8 and Rabin8 mediate Muv formation through FGF secretion in the EGFR-TKI-resistant nematode model. Furthermore, FGFR-TKIs more effectively inhibit the growth of lung cancer cell lines in H1975 (EGFR T790M-L858R; EGFR-TKI-resistant) than H522 (wild-type EGFR) and H1650 (EGFR exon 19 deletion; EGFR-TKI-sensitive) cells, suggesting that FGFR-TKIs could be used to control cancers with EGFR-TKI-resistant mutations.

Analysis of the promoter regions of disease-causing genes in maturity-onset diabetes of the young patients.

Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes caused by the variants in MODY-related genes. In addition to coding variants, variants in the promoter region of MODY-related genes can cause the disease as well. In this study, we screened the promoter regions of the most common MODY-related genes GCK, HNF1A, HNF4A and HNF1B in our cohort of 29 MODY patients. We identified one genetic variant in the HNF1A gene, a 7 bp insertion c.-154-160insTGGGGGT, and three variants in the GCK gene, -282C>T; -194A>G; 402C>G appearing as set. Chloramphenicol acetyltransferase (CAT) assay was performed to test the effect of the 7 bp insertion and the variant set on the activity of the reporter gene in HepG2 and RIN-5F cell, respectively, where a decreasing trend was observed for both variants. In silico analysis and electrophoretic mobility shift assay showed that the 7 bp insertion did not create the binding site for new transcriptional factors, but gave rise to additional binding sites for the existing ones. Results from our study indicated that the 7 bp insertion in the HNF1A gene could be associated with the patient's diabetes. As for the GCK variant set, it is probably not associated with diabetes in patients, but it may modify the fasting glucose level by causing small elevation in variant set carriers. We have presented two promoter variants in MODY-related genes. Variant in the HNF1A gene is presumed to be disease-causing and the GCK promoter variant set could be a phenotype modifier.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome.

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Clinical and Genetic Characterization of 153 Patients with Persistent or Transient Congenital Hyperinsulinism.

CONTEXT: Major advances have been made in the genetics and classification of congenital hyperinsulinism (CHI). OBJECTIVE: To examine the genetics and clinical characteristics of patients with persistent and transient CHI. DESIGN: A cross-sectional study with the register data and targeted sequencing of 104 genes affecting glucose metabolism. PATIENTS: Genetic and phenotypic data were collected from 153 patients with persistent (n = 95) and transient (n = 58) CHI diagnosed between 1972 and 2015. Of these, 86 patients with persistent and 58 with transient CHI participated in the analysis of the selected 104 genes affecting glucose metabolism, including 10 CHI-associated genes, and 9 patients with persistent CHI were included because of their previously confirmed genetic diagnosis. MAIN OUTCOME MEASURES: Targeted next-generation sequencing results and genotype-phenotype associations. RESULTS: Five novel and 21 previously reported pathogenic or likely pathogenic variants in ABCC8, KCNJ11, GLUD1, GCK, HNF4A, and SLC16A1 genes were found in 68% (n = 65) and 0% of the patients with persistent and transient CHI, respectively. KATP channel mutations explained 82% of the mutation positive cases. CONCLUSIONS: The genetic variants found in this nationwide CHI cohort are in agreement with previous studies, mutations in the KATP channel genes being the major causes of the disease. Pathogenic CHI-associated variants were not identified in patients who were both diazoxide responsive and able to discontinue medication within the first 4 months. Therefore, our results support the notion that genetic testing should be focused on patients with inadequate response or prolonged need for medication.

The clinical and genetic characteristics of permanent neonatal diabetes (PNDM) in the state of Qatar.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare condition that occurs within the first six months of life. Permanent NDM (PNDM) is caused by mutations in specific genes that are known for their expression at early and/or late stages of pancreatic beta- cell development, and are either involved in beta-cell survival, insulin processing, regulation, and release. The native population in Qatar continues to practice consanguineous marriages that lead to a high level of homozygosity. To our knowledge, there is no previous report on the genomics of NDM among the Qatari population. The aims of the current study are to identify patients with NDM diagnosed between 2001 and 2016, and examine their clinical and genetic characteristics. METHODS: To calculate the incidence of PNDM, all patients with PNDM diagnosed between 2001 and 2016 were compared to the total number of live births over the 16-year-period. Whole Genome Sequencing (WGS) was used to investigate the genetic etiology in the PNDM cohort. RESULTS: PNDM was diagnosed in nine (n = 9) patients with an estimated incidence rate of 1:22,938 live births among the indigenous Qatari. Seven different mutations in six genes (PTF1A, GCK, SLC2A2, EIF2AK3, INS, and HNF1B) were identified. In the majority of cases, the genetic etiology was part of a previously identified autosomal recessive disorder. Two novel de novo mutations were identified in INS and HNF1B. CONCLUSION: Qatar has the second highest reported incidence of PNDM worldwide. A majority of PNDM cases present as rare familial autosomal recessive disorders. Pancreas associated transcription factor 1a (PTF1A) enhancer deletions are the most common cause of PNDM in Qatar, with only a few previous cases reported in the literature.

Clinical and enzymatic phenotypes in congenital hyperinsulinemic hypoglycemia due to glucokinase-activating mutations: A report of two cases and a brief overview of the literature.

AIMS/INTRODUCTION: The principal aim of this study was to investigate the clinical, genetic and functional characteristics of two cases of congenital hyperinsulinism (CHI) caused by glucokinase (GCK) mutations in young patients. MATERIALS AND METHODS: Novel mutations were detected by CHI next-generation sequencing, and the kinetic parameters and thermal stability of recombinant wild-type and mutant glucokinase were determined in vitro. In addition, 18 naturally occurring GCK-CHI mutations reported previously were also summarized. RESULTS: A de novo mutation (M197V) was found in a 17-year-old male with an epilepsy history, whereas an autosomal dominant mutation (K90R) was found in a 20-year-old female with inherited asymptomatic hypoglycemia. Kinetic analysis showed increased enzyme activity for both mutants (RAI 4.7 for M197V and 1.6 for K90R) and enhanced thermal stability for the M197V mutant. However, of all the GCK-CHI mutants, the increase in enzyme activity (RAI between 1.6 and 130) did not correlate strongly with the severity of hypoglycemia. The de novo group (7/19) showed distinctive phenotypes from the autosomal dominant group (12/19), such as a higher proportion of diazoxide unresponsiveness (28.6% vs 0%), a higher incidence of macrosomia (85.7% vs 40%) and a rarer incidence of adulthood onset (0% vs 25%). CONCLUSIONS: The clinical phenotypes of GCK-CHIs were highly heterogeneous. We have identified two novel GCK-CHI mutations in young patients and investigated their pathogenicity by enzyme kinetic analysis, which expanded the spectrum of this rare disease.

PAQR3 regulates phosphorylation of FoxO1 in insulin-resistant HepG2 cells via NF-κB signaling pathway.

Insulin resistance is a significant feature of type 2 diabetes mellitus and glucose and lipid metabolism disorders. Activation of NF-κB signaling pathway plays an important role in the formation of insulin resistance. FoxO1 plays a major role in regulating glucose and lipid metabolism, as well as insulin signaling pathway. Previous studies have shown that Progestin and AdipoQ Receptor 3 (PAQR3) suppresses the activity of PI3K/Akt, which is an upstream pathway of FoxO1, and additionally promotes the pathological process of diabetic renal inflammatory fibrosis via activating NF-κB pathway. On this basis, it has caused us great concern whether NF-κB is involved in PAQR3 regulation of FoxO1 under insulin resistance. In this study, we aimed to investigate whether PAQR3 regulates phosphorylation of FoxO1 via NF-κB pathway in palmitic acid (PA)-induced insulin-resistant HepG2 cells, thereby causing glucose and lipid metabolism disorders. We found that PA stimulation and PAQR3 overexpression decreased the phosphorylation of FoxO1 and the expressions of glucokinase (GCK) and low density lipoprotein receptor (LDLR), in addition, promoted the nuclear accumulation of NF-κB. Inhibition of NF-κB pathway increased the phosphorylation of FoxO1 and the expressions of GCK and LDLR which were downregulated by PA stimulation and PAQR3 overexpression. Taken together, in PA-induced insulin-resistant HepG2 cells, PAQR3 might regulate the phosphorylation of FoxO1 and the expressions of GCK and LDLR through NF-κB pathway, thereby regulating the glucose and lipid metabolism disorders induced by insulin resistance.