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 characteristics and genetic analysis of children and adolescents with monogenic diabetes].

OBJECTIVE: To explore the clinical characteristics and molecular basis for children and adolescents with monogenic diabetes. METHODS: A retrospective analysis was carried out for the clinical manifestations and laboratory data of 116 children and adolescents diagnosed with diabetes at Ningbo Women and Children's Hospital from January 2020 to March 2023. Whole exome sequencing and mitochondrial gene sequencing were carried out on 21 children with suspected monogenic diabetes. RESULTS: A total of 10 cases of monogenic diabetes were diagnosed, all of which were Maturity-onset Diabetes Of the Young (MODY). Six cases of MODY2 were due to GCK gene mutations, 1 case of MODY3 was due to HNF1A gene mutation, 2 cases of MODY12 were due to ABCC8 gene mutations, and 1 case of MODY13 was due to KCNJ11 gene mutation. Nine of the 10 patients with MODY had no typical symptoms of diabetes. A family history of diabetes was significantly more common in the MODY group compared with the T1DM and T2DM groups (P < 0.05). The BMI of the MODY group was higher than that of the T1DM group (P < 0.05). The initial blood glucose level was lower than that of the T1DM group (P < 0.05), and there was no significant difference compared with the T2DM group. The fasting C-peptide level of the MODY group was higher than that of the T1DM group (P < 0.05), and there was no significant difference compared with the T2DM group. Glycosylated hemoglobin of the MODY group was lower than both the T1DM and T2DM groups (P < 0.05). CONCLUSION: In this study, MODY has accounted for the majority of monogenic diabetes among children and adolescents, and the common mutations were those of the GCK gene in association with MODY2. Blood glucose and glycosylated hemoglobin of children with MODY were slightly increased, whilst the islet cell function had remained, and the clinical manifestations and laboratory tests had overlapped with those of type 2 diabetes. WES and mitochondrial gene sequencing can clarify the etiology of monogenic diabetes and facilitate precise treatment.

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.

Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

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.

Haplotypes of the genes (GCK and G6PC2) underlying the glucose/glucose-6-phosphate cycle are associated with pancreatic beta cell glucose sensitivity in patients with newly diagnosed type 2 diabetes from the VNDS study (VNDS 11).

BACKGROUND: Elevated fasting plasma glucose has been associated with increased risk for development of type 2 diabetes (T2D). The balance between glucokinase (GCK) and glucose-6-phosphate catalytic subunit 2 (G6PC2) activity are involved in glucose homeostasis through glycolytic flux, and subsequent insulin secretion. AIM: In this study, we evaluated the association between the genetic variability of G6PC2 and GCK genes and T2D-related quantitative traits. METHODS: In 794 drug-naïve, GADA-negative, newly diagnosed T2D patients (VNDS; NTC01526720) we performed: genotyping of 6 independent tag-SNPs within GCK gene and 5 tag-SNPs within G6PC2 gene; euglycaemic insulin clamp to assess insulin sensitivity; OGTT to estimate beta-cell function (derivative and proportional control; DC, PC) by mathematical modeling. Genetic association analysis has been conducted using Plink software. RESULTS: Two SNPs within GCK gene (rs882019 and rs1303722) were associated to DC in opposite way (both p < 0.004). Two G6PC2 variants (rs13387347 and rs560887) were associated to both parameters of insulin secretion (DC and PC) and to fasting C-peptide levels (all p < 0.038). Moreover, subjects carrying the A allele of rs560887 showed higher values of 2h-plasma glucose (2hPG) (p = 0.033). Haplotype analysis revealed that GCK (AACAAA) haplotype was associated to decreased fasting C-peptide levels, whereas, the most frequent haplotype of G6PC2 (GGAAG) was associated with higher fasting C-peptide levels (p = 0.001), higher PC (ß = 6.87, p = 0.022) and the lower 2hPG (p = 0.012). CONCLUSION: Our findings confirmed the role of GCK and G6PC2 in regulating the pulsatility in insulin secretion thereby influencing insulin-signaling and leading to a gradual modulation in glucose levels in Italian patients with newly diagnosed T2D.

The C7orf43/TRAPPC14 component links the TRAPPII complex to Rabin8 for preciliary vesicle tethering at the mother centriole during ciliogenesis.

The primary cilium is a cellular sensor that detects light, chemicals, and movement and is important for morphogen and growth factor signaling. The small GTPase Rab11-Rab8 cascade is required for ciliogenesis. Rab11 traffics the guanine nucleotide exchange factor (GEF) Rabin8 to the centrosome to activate Rab8, needed for ciliary growth. Rabin8 also requires the transport particle protein complex (TRAPPC) proteins for centrosome recruitment during ciliogenesis. Here, using an MS-based approach for identifying Rabin8-interacting proteins, we identified C7orf43 (also known as microtubule-associated protein 11 (MAP11)) as being required for ciliation both in human cells and zebrafish embryos. We find that C7orf43 directly binds to Rabin8 and that C7orf43 knockdown diminishes Rabin8 preciliary centrosome accumulation. Interestingly, we found that C7orf43 co-sediments with TRAPPII complex subunits and directly interacts with TRAPPC proteins. Our findings establish that C7orf43 is a TRAPPII-specific complex component, referred to here as TRAPPC14. Additionally, we show that TRAPPC14 is dispensable for TRAPPII complex integrity but mediates Rabin8 association with the TRAPPII complex. Finally, we demonstrate that TRAPPC14 interacts with the distal appendage proteins Fas-binding factor 1 (FBF1) and centrosomal protein 83 (CEP83), which we show here are required for GFP-Rabin8 centrosomal accumulation, supporting a role for the TRAPPII complex in tethering preciliary vesicles to the mother centriole during ciliogenesis. In summary, our findings have revealed an uncharacterized TRAPPII-specific component, C7orf43/TRAPPC14, that regulates preciliary trafficking of Rabin8 and ciliogenesis and support previous findings that the TRAPPII complex functions as a membrane tether.

Pathogenicity of a glucokinase gene mutation and description of its clinical phenotype.

Glucokinase gene (GCK) mutations comprise approximately 10% of cases of maturity-onset diabetes of the young (MODY). Over 800 different mutations in GCK have been reported in the Human Gene Mutation Database, the vast majority of which result in MODY type 2. The missense mutation p.Leu122Val is listed in that database as "disease-causing;" however, the National Center for Biotechnology Information ClinVar database (Variation ID 585919) reports that this mutation is of "uncertain significance." Both databases reference the same Italian pediatric patient reported by Massa et al in 2001, but no phenotypic description of the patient is included in the original article. We report a pedigree of three patients over two generations affected with GCK mutation c.364C > G (p.Leu122Val) to support the clinical significance of this mutation and to provide the first phenotypic description of patients with this particular mutation.

Next generation sequencing targeted gene panel in Greek MODY patients increases diagnostic accuracy.

BACKGROUND: Maturity Onset Diabetes of the Young (MODY) constitutes a genetically and clinically heterogeneous type of monogenic diabetes. It is characterized by early onset, autosomal dominant inheritance and a defect in pancreatic ß-cell insulin secretion. To date, various MODY subtypes have been reported, each one of a distinct genetic etiology. OBJECTIVE: The aim of this study was to identify the molecular defects of 50 patients with MODY employing the methodology of next generation sequencing (NGS) targeted gene panel. METHODS: A panel of seven MODY genes was designed and employed to screen 50 patients fulfilling the MODY diagnostic criteria. Patients with no pathogenic, likely pathogenic or uncertain significance variants detected, were further tested by multiplex ligation-dependent probe amplification (MLPA) for copy number variations (CNVs). RESULTS: Eight different pathogenic or likely pathogenic variants were identified in eight MODY patients (diagnostic rate 16%). Five variants of uncertain significance were also detected in seven MODY patients. Five novel pathogenic and likely pathogenic variants were detected in the genes GCK; p.Cys371X, HNF1A; p.Asn402Tyr, HNF4A; p.Glu285Lys, and ABCC8; p.Met1514Thr and p.Ser1386Phe. Two de novo heterozygous deletions of the entire HNF1B gene were detected in two patients, raising the diagnostic rate to 20%. CONCLUSIONS: Although many MODY patients still remain without exact MODY type identification, the application of NGS methodology provided rapid results, increased diagnostic accuracy, and was cost-effective compared to Sanger sequencing. Accurate genetic diagnosis of the MODY subtype is important for treatment selection, disease prognosis, and family counseling.

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.

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.

Association of GCK gene DNA methylation with the risk of clopidogrel resistance in acute coronary syndrome patients.

BACKGROUNDS: Clopidogrel resistance (CR), which was manifested as the failure of platelet inhibition in clopidogrel treatment, was likely to lead to cardiovascular events. Our study was aimed to explore the contribution of DNA methylation in glucokinase (GCK) to the CR risk. METHODS: Among 36 CR and 36 non-CR acute coronary syndrome (ACS) patients, the platelet functions were evaluated by VerifyNow P2Y12 assay (turbidimetric-based optical detection) and DNA methylation levels on two fragments of the CGI from the GCK were investigated through bisulfite pyrosequencing methods. In addition, the GCK mRNA expression was analyzed via quantitative real-time PCR. Lastly, the logistic regression was employed to test the interaction between GCK methylation and nongenetic variables in CR patients. RESULTS: Subunit analysis showed that in male patients without DM but suffering from dyslipidemia, the increased methylation of cg18492943 indicated a risk of poor clopidogrel response (male, NCR vs CR(%): 84.86 ± 6.29 vs 88.16 ± 4.32, P = .032; without DM, NCR vs CR (%): 84.66 ± 6.18 vs 88.16 ± 4.17, P = .029; and dyslipidemia, NCR vs CR (%): 83.81 ± 6.96 vs 88.39 ± 4.74, P = .042).In addition, GCK mRNA expression was reduced in CR patients without DM. Moreover, regression analysis indicated that the values of platelet distribution width (PDW), total cholesterol (TC), and uric acid (UA) were correlated with the incidence of CR, and hypertension lowered the CR risk. CONCLUSIONS: A higher methylation of cg18492943 in GCK gene would lower the expression of GCK mRNA, which might contribute to CR in patients without DM. Meanwhile, PDW and TC might be risk factors in CR.

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.

Congenital diabetes mellitus.

Congenital diabetes mellitus is a rare disorder characterized by hyperglycemia that occurs shortly after birth. We define "Diabetes of Infancy" if hyperglycemia onset before 6 months of life. From the clinical point of view, we distinguish two main types of diabetes of infancy: transient (TNDM), which remits spontaneously, and permanent (PNDM), which requires lifelong treatment. TNDM may relapse later in life. About 50% of cases are transient (TNDM) and 50% permanent. Clinical manifestations include severe intrauterine growth retardation, hyperglycemia and dehydration. A wide range of different associated clinical signs including facial dysmorphism, deafness and neurological, cardiac, kidney or urinary tract anomalies are reported. Developmental delay and learning difficulties may also be observed. In this paper we review all the causes of congenital diabetes and all genes and syndromes involved in this pathology. The discovery of the pathogenesis of most forms of congenital diabetes has made it possible to adapt the therapy to the diagnosis and in the forms of alteration of the potassium channels of the pancreatic Beta cells the switch from insulin to glibenclamide per os has greatly improved the quality of life. Congenital diabetes, although it is a very rare form, has been at the must of research in recent years especially for pathogenesis and pharmacogenetics. The most striking difference compared to the more frequent autoimmune diabetes in children (type 1 diabetes) is the possibility of treatment with hypoglycemic agents and the apparent lower frequency of chronic complications.

Recombinant protein CCN5/WISP2 promotes islet cell proliferation and survival in vitro.

Pancreatic ß cell proliferation, survival and function are key elements that need to be considered in developing novel antidiabetic therapies. We recently identified CCN5/WISP2 to have potential growth promoting properties when overexpressed in ß cells; however, further investigations are needed to validate those properties. In this study, we demonstrated that exogenous treatment of insulinoma cells and primary islets with recombinant CCN5 (rh-CCN5) protein enhanced the proliferative capacity which was correlated with activation of cell-cycle regulators CDK4 and cyclin D1. Furthermore, pre-incubation of these cells with rh-CCN5 enhanced their survival rate after being exposed to harsh treatments such as streptozotocin and high concentrations of glucose and free fatty acids. CCN5 as well caused an upregulation in the expression of key genes associated with ß cell identity and function such as GLUT-2 and GCK. Finally, CCN5 activated FAK and downstream ERK kinases which are known to stimulate cell proliferation and survival. Hence, our results validate the growth promoting activities of rh-CCN5 in ß cells and open the door for further investigations in vivo.

Heterozygous lys169Glu mutation of glucokinase gene in a Chinese family having glucokinase-maturity-onset diabetes of the young (GCK-MODY).

We report a 24-year-old female with early-onset and persistent mild fasting hyperglycemia due to glucokinase-maturity-onset diabetes of the young (GCK-MODY). A c.505A>G (p. Lys169Glu) missense mutation of the GCK gene was identified. In silico analysis indicated that the mutation affected a conserved amino acid and is disease-causing. This report describes GCK-MODY in a Chinese family and stresses that in managing this condition it is important to avoid unnecessary drug treatment and excessive anxiety about mild hyperglycemia.

Development of a clinical calculator to aid the identification of MODY in pediatric patients at the time of diabetes diagnosis.

Maturity Onset Diabetes of the Young (MODY) is a young-onset, monogenic form of diabetes without needing insulin treatment. Diagnostic testing is expensive. To aid decisions on who to test, we aimed to develop a MODY probability calculator for paediatric cases at the time of diabetes diagnosis, when the existing "MODY calculator" cannot be used. Firth logistic regression models were developed on data from 3541 paediatric patients from the Swedish 'Better Diabetes Diagnosis' (BDD) population study (n = 46 (1.3%) MODY (HNF1A, HNF4A, GCK)). Model performance was compared to using islet autoantibody testing. HbA1c, parent with diabetes, and absence of polyuria were significant independent predictors of MODY. The model showed excellent discrimination (c-statistic = 0.963) and calibrated well (Brier score = 0.01). MODY probability > 1.3% (ie. above background prevalence) had similar performance to being negative for all 3 antibodies (positive predictive value (PPV) = 10% v 11% respectively i.e. ~ 1 in 10 positive test rate). Probability > 1.3% and negative for 3 islet autoantibodies narrowed down to 4% of the cohort, and detected 96% of MODY cases (PPV = 31%). This MODY calculator for paediatric patients at time of diabetes diagnosis will help target genetic testing to those most likely to benefit, to get the right diagnosis.

Whole Exome Sequencing in Children With Type 1 Diabetes Before Age 6 Years Reveals Insights Into Disease Heterogeneity.

Aims: This study is aimed at comparing whole exome sequencing (WES) data with the clinical presentation in children with type 1 diabetes onset ≤ 5 years of age (EOT1D). Methods: WES was performed in 99 unrelated children with EOT1D with subsequent analysis to identify potentially deleterious rare variants in MODY genes. High-resolution HLA class II haplotyping, SNP genotyping, and T1D-genetic risk score (T1D-GRS) were also evaluated. Results: Eight of the ninety-nine EOT1D participants carried a potentially deleterious rare variant in a MODY gene. Rare variants affected five genes: GCK (n = 1), HNF1B (n = 2), HNF4A (n = 1), PDX1 (n = 2), and RFX6 (n = 2). At diagnosis, these children had a mean age of 3.0 years, a mean HbA1c of 10.5%, a detectable C-peptide in 5/8, and a positive islet autoantibody in 6/7. Children with MODY variants tend to exhibit a lower number of pancreatic autoantibodies and a lower fasting C-peptide compared to EOT1D without MODY rare variants. They also carried at least one high-risk DR3-DQ2 or DR4-DQ8 haplotype and exhibited a T1D-GRS similar to the other individuals in the EOT1D cohort, but higher than healthy controls. Conclusions: WES found potentially deleterious rare variants in MODY genes in 8.1% of EOT1D, occurring in the context of a T1D genetic background. Such genetic variants may contribute to disease precipitation by a ß-cell dysfunction mechanism. This supports the concept of different endotypes of T1D, and WES at T1D onset may be a prerequisite for the implementation of precision therapies in children with autoimmune diabetes.

NDR kinase tricornered genetically interacts with Ccm3 and metabolic enzymes in Drosophila melanogaster tracheal development.

The Germinal Center Kinase III (GckIII) pathway is a Hippo-like kinase module defined by sequential activation of Ste20 kinases Thousand and One (Tao) and GckIII, followed by nuclear dbf2-related (NDR) kinase Tricornered (Trc). We previously uncovered a role for the GckIII pathway in Drosophila melanogaster tracheal (respiratory) tube morphology. The trachea form a network of branched epithelial tubes essential for oxygen transport, and are structurally analogous to branched tubular organs in vertebrates, such as the vascular system. In the absence of GckIII pathway function, aberrant dilations form in tracheal tubes characterized by mislocalized junctional and apical proteins, suggesting that the pathway is important in maintaining tube integrity in development. Here, we observed a genetic interaction between trc and Cerebral cavernous malformations 3 (Ccm3), the Drosophila ortholog of a human vascular disease gene, supporting our hypothesis that the GckIII pathway functions downstream of Ccm3 in trachea, and potentially in the vertebrate cerebral vasculature. However, how GckIII pathway signaling is regulated and the mechanisms that underpin its function in tracheal development are unknown. We undertook biochemical and genetic approaches to identify proteins that interact with Trc, the most downstream GckIII pathway kinase. We found that known GckIII and NDR scaffold proteins are likely to control GckIII pathway signaling in tracheal development, consistent with their conserved roles in Hippo-like modules. Furthermore, we show genetic interactions between trc and multiple enzymes in glycolysis and oxidative phosphorylation, suggesting a potential function of the GckIII pathway in integrating cellular energy requirements with maintenance of tube integrity.