Presence of immunogenic alternatively spliced insulin gene product in human pancreatic delta cells.

AIMS/HYPOTHESIS: Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. METHODS: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1ß release. RESULTS: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. CONCLUSIONS/INTERPRETATION: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. DATA AVAILABILITY: The full EM dataset is available via www.nanotomy.org (for review: http://www.nanotomy.org/OA/Tienhoven2021SUB/6126-368/ ). Single-cell RNA-seq data was made available by Segerstolpe et al [13] and can be found at https://sandberglab.se/pancreas . The RNA and protein sequence of INS-splice was uploaded to GenBank (BankIt2546444 INS-splice OM489474).

Disrupting methyl-CpG-binding protein 2 expression induces the transformation of induced pluripotent stem cell cardiomyocytes into pacemaker-like cells by insulin gene enhancer binding protein 1.

BACKGROUND: The experiment will explore whether interfering with the expression of methyl-CpG-binding protein 2 (MecP2) can enhance the ability of insulin gene enhancer binding protein 1 (ISL1) to induce iPSC-CMs to differentiate into pacemaker-like cells. METHODS: Differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes (CMs) can be induced via the regulation of the Wnt signaling pathway. Real-time quantitative PCR (qPCR), western blotting, immunofluorescence staining, and patch-clamp technique were used to analyze the ability of ISL1 to induce the transformation of iPSC-CMs into pacemaker-like cells. Calcium spark, patch-clamp technique, and real-time qPCR were used to verify whether disrupting the expression of MeCP2 enhanced this ability of ISL1 to induce the differentiation of iPSC-CMs into pacemaker cells. Transplant pacemaker-like cardiomyocytes into the myocardium of mice to observe whether the pacemaker cells can survive in the tissue for a long time. RESULTS: RT-qPCR and patch-clamp analyses showed that overexpression of ISL1 induced the successful differentiation of iPSC-CMs into pacemaker cells. ISL1-overexpressing pacemaker-like cells possessed typical characteristics of pacemaker morphology, including action potential and If inward current. Chromatin immunoprecipitation results showed that MeCP2 bound to the promoter region of HCN4. Following disruption of MeCP2 expression, the gene expression of sinoatrial node-specific transcription factors, If inward current, and cardiac rhythm changes in iPSC-CMs resembled those of sinoatrial node pacemaker cells. Therefore, ISL1 induced the differentiation of iPSC-CMs into pacemaker-like cells, and knockdown of MeCP2 increased this effect. Frozen section results showed that surviving pacemaker-like cells could still be observed in myocardial tissue after 45 days. CONCLUSIONS: Experiments have found that interfering with the expression of MeCP2 can increase the ability of ISL1 to induce iPSC-CM cells to differentiate into pacemaker-like cells. And the pacemaker-like cells obtained in this experiment can survive in myocardial tissue for a long time.

Effect of chronic exposure to ketohexoses on pancreatic ß-cell function in INS-1 rat insulinoma cells.

Glucotoxicity, impaired insulin secretion, suppression of insulin gene expression, and apoptosis, in pancreatic ß-cells caused by chronic hyperglycemia is a key component of the pathogenesis of type 2 diabetes. Recently, it has been reported that rare sugar d-allulose has antihyperglycemic and antihyperlipidemic effects in diabetic rats. However, the direct effects of rare sugars including d-allulose on pancreatic ß-cell function are unclear. In this study, we investigated whether chronic exposure to ketohexoses causes glucotoxicity, suppression of insulin gene expression, and apoptosis, in INS-1 rat pancreatic insulinoma cells. d-Fructose, d-tagatose, l-allulose, and l-sorbose treatment for 1-week reduced insulin gene expression, whereas d-allulose, d-sorbose, l-fructose, and l-tagatose did not. All ketohexoses were transported into INS-1 cells, but were not metabolized. In addition, the ketohexoses did not induce apoptosis and did not affect glucose metabolism. These results suggest that long-term administration of d-allulose, d-sorbose, l-fructose, and l-tagatose does not affect pancreatic ß-cell function.

Generation of a Beta-Cell Transplant Animal Model of Diabetes Using CRISPR Technology.

Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.

Bone marrow-derived mesenchymal stem cells improve rat islet graft revascularization by upregulating ISL1.

Revascularization of the islet transplant is a crucial step that defines the success rate of patient recovery. Bone marrow-derived mesenchymal stem cells (BMSCs) have been reported to promote revascularization; however, the underlying cellular mechanism remains unclear. Moreover, our liquid chromatography-tandem mass spectrometry results showed that BMSCs could promote the expression of insulin gene enhancer binding protein-1 (ISL1) in islets. ISL1 is involved in islets proliferation and plays a potential regulatory role in the revascularization of islets. This study identifies the ISL1 protein as a potential modulator in BMSCs-mediated revascularization of islet grafts. We demonstrated that the survival rate and insulin secretion of islets were increased in the presence of BMSCs, indicating that BMSCs promote islet revascularization in a coculture system and rat diabetes model. Interestingly, we also observed that the presence of BMSCs led to an increase in ISL1 and vascular endothelial growth factor A (VEGFA) expression in both islets and the INS-1 rat insulinoma cell line. In silico protein structure modeling indicated that ISL1 is a transcription factor that has four binding sites with VEGFA mRNA. Further results showed that overexpression of ISL1 increased both the abundance of VEGFA transcripts and protein accumulation, while inhibition of ISL1 decreased the abundance of VEGFA. Using a ChIP-qPCR assay, we demonstrated that direct molecular interactions between ISL1 and VEGFA occur in INS-1 cells. Together, these findings reveal that BMSCs promote the expression of ISL1 in islets and lead to an increase in VEGFA in islet grafts. Hence, ISL1 is a potential target to induce early revascularization in islet transplantation.

Identification of Ala2Thr mutation in insulin gene from a Chinese MODY10 family.

More than 80% of maturity-onset diabetes of the young (MODY) in Chinese is genetically unexplained. To investigate whether the insulin gene (INS) mutation is responsible for some Chinese MODY, we screened INS mutations causing MODY10 in MODY pedigrees and explored the potential pathogenic mechanisms. INS mutations were screened in 56 MODY familial probands. Structure-function characterization and clinical profiling of identified INS mutations were conducted. An INS mutation, at the position 2 alanine-to-threonine substitution (A2T), was identified and co-segregated with hyperglycemia in a MODY pedigree. The A2T mutation converted an α-helix into a ß-sheet at the N-terminal of the signal peptide (SP) of preproinsulin. The A2T mutation did not affect preproinsulin translocation across endoplasmic reticulum (ER) membrane, but impaired its SP cleavage within the ER. In INS-1 cells transfected with an A2T mutant, glucose-stimulated insulin secretion (GSIS) was significantly decreased, while BiP luciferase activities were significantly increased compared to that of wild type (WT). We identified an INS-A2T mutation cosegregating with diabetes in a Chinese MODY pedigree. This mutation severely impaired SP cleavage and thus blocked the formation of proinsulin, resulting in enhanced ER stress, which may be responsible for decreased insulin secretion and subsequently, the onset of MODY10.

[Alternative Variants of Pax4 Human Transcription Factor: Comparative Transcriptional Activity].

MODY is a group of genetically and clinically heterogeneous forms of diabetes characterized by auto-somal dominant inheritance and is subdivided in 13 subtypes dependent on the gene involved. The subtype MODY9 is a very rare form caused by mutations in the gene encoding the PAX4 transcription factor which is engaged in differentiation of pancreatic beta-cells. PAX4 contains two DNA-binding domains-Paired and Homeo. Expression of the human PAX4 gene is tissue-specific. The alternatively spliced mRNA variants encode for protein isoforms which differ within their N- and C-terminal regions. In this study, the transcriptional activities of the human PAX4 variants, both known and new ones, were determined. The full-length PAX4 containing intact DNA-binding domains was found to have maximal activity in transient expression system of the firefly luciferase reporter gene under control of the insulin promoter in HEK293 cells. The transcriptional activity is significantly reduced in the variants lacking eight N-terminal amino acid residues and/or variants whose Homeo domain is truncated from the C-terminus. Similar data were obtained with the glucagon promoter reporter system. The aberrant PAX4 variants were shown to retain stability and nuclear localization.

Allele-specific methylation of type 1 diabetes susceptibility genes.

The susceptibility to autoimmune diseases is influenced by genes encoding major histocompatibility complex (MHC) proteins. By examining the epigenetic methylation maps of cord blood samples, we found marked differences in the methylation status of CpG sites within the MHC genes (cis-metQTLs) between carriers of the type 1 diabetes risk haplotypes HLA-DRB1*03-DQA1*0501-DQB1*0201 (DR3-DQ2) and HLA-DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8). These differences were found in children and adults, and were accompanied by reduced HLA-DR protein expression in immune cells with the HLA-DR3-DQ2 haplotype. Extensive cis-metQTLs were identified in all 45 immune and non-immune type 1 diabetes susceptibility genes analyzed in this study. We observed and validated a novel association between the methylation status of CpG sites within the LDHC gene and the development of insulin autoantibodies in early childhood in children who are carriers of the highest type 1 diabetes risk genotype. Functionally relevant epigenetic changes in susceptibility genes may represent therapeutic targets for type 1 diabetes.

A novel INS mutation in a family with maturity-onset diabetes of the young: Variable insulin secretion and putative mechanisms.

Insulin gene (INS) mutations cause a rare form of maturity-onset diabetes of the young (MODY), a heterogeneous group of autosomal dominant diabetes with at least 14 confirmed causative genes. Here, we describe a family with MODY due to a novel INS mutation, detected using massively parallel sequencing (MPS). The proband presented aged 11 years with mild diabetic ketoacidosis. She was negative for IA2 and GAD antibodies. She had a strong family history of diabetes affecting both her two siblings and her mother, none of whom had ketosis but who were considered to have type 1 diabetes and managed on insulin, and her maternal grandfather, who was managed for decades on sulfonylureas. Of note, her younger sister had insulin deficiency but an elevated fasting proinsulin:insulin ratio of 76% (ref 5%-30%). Sanger sequencing of HNF4A, HNF1A, and HNF1B in the proband was negative. Targeted MPS using a custom-designed amplicon panel sequenced on an Illumina MiSeq detected a heterozygous INS mutation c.277G>A (p.Glu93Lys). Sanger sequencing confirmed the variant segregated with diabetes within the family. Structural analysis of this variant suggested disruption of a critical hydrogen bond between insulin and the insulin receptor; however, the clinical picture in some individuals also suggested abnormal insulin processing and insulin deficiency. This family has a novel INS mutation and demonstrated variable insulin deficiency. MPS represents an efficient method of MODY diagnosis in families with rarer gene mutations.

In vivo measurement and biological characterisation of the diabetes-associated mutant insulin p.R46Q (GlnB22-insulin).

AIMS/HYPOTHESIS: Heterozygous mutations in the insulin gene that affect proinsulin biosynthesis and folding are associated with a spectrum of diabetes phenotypes, from permanent neonatal diabetes to MODY. In vivo studies of these mutations may lead to a better understanding of insulin mutation-associated diabetes and point to the best treatment strategy. We studied an 18-year-old woman with MODY heterozygous for the insulin mutation p.R46Q (GlnB22-insulin), measuring the secretion of mutant and wild-type insulin by LC-MS. The clinical study was combined with in vitro studies of the synthesis and secretion of p.R46Q-insulin in rat INS-1 insulinoma cells. METHODS: We performed a standard 75 g OGTT in the 18-year-old woman and measured plasma glucose and serum insulin (wild-type insulin and GlnB22-insulin), C-peptide, proinsulin, glucagon and amylin. The affinity of GlnB22-insulin was tested on human insulin receptors expressed in baby hamster kidney (BHK) cells. We also examined the subcellular localisation, secretion and impact on cellular stress markers of p.R46Q-insulin in INS-1 cells. RESULTS: Plasma GlnB22-insulin concentrations were 1.5 times higher than wild-type insulin at all time points during the OGTT. The insulin-receptor affinity of GlnB22-insulin was 57% of that of wild-type insulin. Expression of p.R46Q-insulin in INS-1 cells was associated with decreased insulin secretion, but not induction of endoplasmic reticulum stress. CONCLUSIONS/INTERPRETATION: The results show that beta cells can process and secrete GlnB22-insulin both in vivo and in vitro. Our combined approach of immunoprecipitation and LC-MS to measure mutant and wild-type insulin may be useful for the study of other mutant insulin proteins. The ability to process and secrete a mutant protein may predict a more benign course of insulin mutation-related diabetes. Diabetes develops when the beta cell is stressed because of increased demand for insulin, as observed in individuals with other insulin mutations that affect the processing of proinsulin to insulin or mutations that reduce the affinity for the insulin receptor.

Venom Insulins of Cone Snails Diversify Rapidly and Track Prey Taxa.

A specialized insulin was recently found in the venom of a fish-hunting cone snail, Conus geographus Here we show that many worm-hunting and snail-hunting cones also express venom insulins, and that this novel gene family has diversified explosively. Cone snails express a highly conserved insulin in their nerve ring; presumably this conventional signaling insulin is finely tuned to the Conus insulin receptor, which also evolves very slowly. By contrast, the venom insulins diverge rapidly, apparently in response to biotic interactions with prey and also possibly the cones' own predators and competitors. Thus, the inwardly directed signaling insulins appear to experience predominantly purifying sele\ction to target an internal receptor that seldom changes, while the outwardly directed venom insulins frequently experience directional selection to target heterospecific insulin receptors in a changing mix of prey, predators and competitors. Prey insulin receptors may often be constrained in ways that prevent their evolutionary escape from targeted venom insulins, if amino-acid substitutions that result in escape also degrade the receptor's signaling functions.

Genetic characteristics, clinical spectrum, and incidence of neonatal diabetes in the Emirate of AbuDhabi, United Arab Emirates.

Neonatal diabetes mellitus (NDM) can be transient (TNDM) or permanent (PNDM). Data on NDM from the Gulf region are limited to few studies on PNDM.The objective of this study was to describe the genetic and clinical spectrum of NDM and estimate its incidence in AbuDhabi, capital of the United Arab Emirate (UAE). Patients were identified from the pediatric diabetes clinics and sequencing of known NDM genes was conducted in all families. Twenty-five patients were identified. Incidence during 1985-2013 was 1:29,241 Live births. Twenty-three out of twenty-five had PNDM (incidence 1:31,900) and 2/25 had TNDM (incidence 1:350,903). Eleven out of twenty-five had extra-pancreatic features and three had pancreatic aplasia. The genetic cause was detected in 21/25 (84%). Of the PNDM patients, nine had recessive EIF2AK3 mutations, six had homozygous INS mutations, two with deletion of the PTF1A enhancer, one was heterozygous for KCNJ11 mutation, one harboured a novel ABCC8 variant, and 4/21 without mutations in all known PNDM genes. One TNDM patient had a 6q24 methylation defect and another was homozygous for the INS c-331C>G mutation. This mutation also caused permanent diabetes with variable age of onset from birth to 18 years. The parents of a child with Wolcott-Rallison syndrome had a healthy girl following pre-implantation genetic diagnosis. The child with KCNJ11 mutation was successfully switched from insulin to oral sulphonylurea. The incidence of PNDM in Abu Dhabi is among the highest in the world and its spectrum is different from Europe and USA. In our cohort, genetic testing has significant implications for the clinical management.

Bilateral cataracts in a 6-yr-old with new onset diabetes: a novel presentation of a known INS gene mutation.

The prevalence of diabetes-related cataracts during childhood is less than 1%. When cataracts occur, it is often in adolescent females with prolonged symptoms and significant hyperglycemia. Cataracts are not a classic feature of monogenic diabetes. We report a case of a 6-yr-old, previously healthy Caucasian male, who presented with bilateral acquired cataracts and was subsequently diagnosed with new onset diabetes. Additional symptoms at presentation included a several year history of polyuria and polydipsia, mild hepatomegaly, and short stature. Pertinent negatives include acanthosis nigricans, lipoatrophy, deafness, muscle weakness, or neuropathy. HbA1c was significantly elevated at diagnosis (>14%, 129.5 mmol/mol) without evidence of ketosis. Autoantibody testing was negative. Features of Mauriac syndrome (short stature, hepatomegaly) as well as acquired cataracts indicated long-standing hyperglycemia with sufficient insulin production to prevent ketone production and development of diabetic ketoacidosis. Whole exome sequencing was conducted and a de novo heterozygous mutation in the INS gene (c.94G>A; p.Gly32Ser) was identified. INS gene mutations are common causes of permanent neonatal diabetes but rare causes of antibody-negative diabetes in children. Importantly, INS gene mutations have not been previously associated with acquired cataracts. Knowledge of a monogenic cause of diabetes allows clinicians to tailor counseling and screening of diabetes-related comorbidities. In summary, this case highlights the need to consider testing for monogenic diabetes, specifically INS gene mutations, in pediatric patients with antibody-negative diabetes, especially if complications of prolonged hyperglycemia are present at diagnosis.

Permanent neonatal diabetes in siblings with novel C109Y INS mutation transmitted by an unaffected parent with somatic mosaicism.

Mutations involving the insulin (INS) gene are a common cause of permanent neonatal diabetes (PND). Although INS mutations typically occur de novo and germline INS mutations transmitted to offspring by unaffected parents has been described, somatic mosaicism in a parent with an INS mutation has not been previously reported. We describe two siblings (one brother and one sister) with PND (26- and 19-yr old diagnosed at 3 and 7 months old, respectively), whose parents were unaffected. We performed genetic analysis of leukocyte DNA for this family. Both patients were found to carry the novel heterozygous c.326G>A substitution in exon 3 of INS, resulting in a p.C109Y change of the insulin protein. Analyses of leukocyte DNA from the parents revealed low level mutation in the sequencing trace of the father, raising the possibility of somatic mosaicism. Real-time polymerase chain reaction (PCR) analysis showed he had approximately 73% of the mutant allele relative to his affected son. This first report of somatic mosaicism in an unaffected parent with an INS mutation suggests that parental mosaicism may be responsible for the transmission of PND in patients with de novo INS mutations. As such, appropriate counseling for recurrent risks should be considered and we recommend that molecular genetic testing for future siblings at birth should be offered to the parents of children with INS mutation.

Novel Approach for Treating Diabetes in a Patient With the Heterozygous Pathogenic Variant R46Q in the Insulin Gene.

Maturity-onset diabetes of the young (MODY) is a monogenic disorder of glucose homeostasis with several subtypes, each defined by a distinct genetic etiology. Heterozygous pathogenic variants in the insulin gene are rare causes of MODY, and optimal treatment strategies remain uncertain. Herein we describe a patient with diabetes caused by the heterozygous pathogenic variant R46Q in the insulin gene and the glycemic response to selected antidiabetic treatment regimens. The R46Q pathogenic variant leads to secretion of both mutant and wild-type insulin. In vitro, the mutant insulin is associated with a lower insulin-receptor affinity compared with wild-type insulin and a decline in wild-type insulin secretion. In our patient, treatment with a combination of long- and short-acting insulin led to a decline in hemoglobin A1C (HbA1c), although not to the recommended target. A shift to metformin and subsequent add-on of a sodium-glucose cotransporter 2 inhibitor (SGLT2i) resulted in HbA1c levels of less than 7% (53 mmol/mol) and durable glycemic control. Continuous glucose monitoring and oral glucose tolerance tests confirmed that treatment with metformin and SGLT2i was superior to treatment with insulin. In conclusion, diabetes caused by the pathogenic variant R46Q in the insulin gene may be effectively treated with noninsulin.

Clinical, glycometric features and treatment in a family with monogenic diabetes due to a new mutation in the insulin gene.

Monogenic diabetes caused by changes in the gene that encodes insulin (INS) is a very rare form of monogenic diabetes (<1%). The aim of this work is to describe the clinical and glycaemic control characteristics over time from four members of a family diagnosed with monogenic diabetes with the novel mutation: c.206del,p.(Gly69Aalfs*62) located in exon 3 of the gene INS. 75% are females, with debut in adolescence and negative autoimmunity. In all cases, C-peptide is detectable decades after diagnosis (>0.6ng/ml). Currently, patients are being treated either with insulin in a bolus-basal regimen, oral antidiabetics or hybrid closed loop system. Monogenic diabetes due to mutation in the INS is an entity with heterogeneous presentation, whose diagnosis requires high suspicion and presents an important clinical impact. Given the lack of standards in this regard, therapy must be individualized, although insulin therapy could help preserve beta cell functionality in these subjects.

A Review of the Biosynthesis and Structural Implications of Insulin Gene Mutations Linked to Human Disease.

The discovery of the insulin hormone over 100 years ago, and its subsequent therapeutic application, marked a key landmark in the history of medicine and medical research. The many roles insulin plays in cell metabolism and growth have been revealed by extensive investigations into the structure and function of insulin, the insulin tyrosine kinase receptor (IR), as well as the signalling cascades, which occur upon insulin binding to the IR. In this review, the insulin gene mutations identified as causing disease and the structural implications of these mutations will be discussed. Over 100 studies were evaluated by one reviewing author, and over 70 insulin gene mutations were identified. Mutations may impair insulin gene transcription and translation, preproinsulin trafficking and proinsulin sorting, or insulin-IR interactions. A better understanding of insulin gene mutations and the resultant pathophysiology can give essential insight into the molecular mechanisms underlying impaired insulin biosynthesis and insulin-IR interaction.

Methylation haplotypes of the insulin gene promoter in children and adolescents with type 1 diabetes: Can a dimensionality reduction approach predict the disease?

DNA methylation of cytosine-guanine sites (CpGs) is associated with type 1 diabetes (T1D). The sequence of methylated and non-methylated sites in a specific genetic region constitutes its methyl-haplotype. The aim of the present study was to identify insulin gene promoter (IGP) methyl-haplotypes among children and adolescents with T1D and suggest a predictive model for the discrimination of cases and controls according to methyl-haplotypes. A total of 40 individuals (20 T1D) participated. The IGP region from peripheral whole blood DNA of 40 participants (20 T1D) was sequenced using next-generation sequencing, sequences were read using FASTQ files and methylation status was calculated by python-based pipeline for targeted deep bisulfite sequenced amplicons (ampliMethProfiler). Methylation profile at 10 CpG sites proximal to transcription start site of the IGP was recorded and coded as 0 for unmethylation or 1 for methylation. A single read could result in '1111111111' methyl-haplotype (all methylated), '000000000' methyl-haplotype (all unmethylated) or any other combination. Principal component analysis was applied to the generated methyl-haplotypes for dimensionality reduction, and the first three principal components were employed as features with five different classifiers (random forest, decision tree, logistic regression, Naive Bayes, support vector machine). Naive Bayes was the best-performing classifier, with 0.9 accuracy. Predictive models were evaluated using receiver operating characteristics (AUC 0.96). Methyl-haplotypes '1111111111', '1111111011', '1110111111', '1111101111' and '1110101111' were revealed to be the most significantly associated with T1D according to the dimensionality reduction method. Methylation-based biomarkers such as IGP methyl-haplotypes could serve to identify individuals at high risk for T1D.

Maturity-onset diabetes of the young type 10 caused by an Ala2Thr mutation of INS: A case report.

BACKGROUND: Maturity-onset diabetes of the young 10 caused by the c.4G>A (p.Ala2Thr) mutation is extremely rare, with only two reported studies to date. Herein, we report another case that differs from previous cases in phenotype. CASE SUMMARY: The proband developed diabetes at the age of 27 years, despite having a normal body mass index (BMI). She exhibited partial impairment of islet function, tested positive for islet antibodies, and required high doses of insulin. Her sister also carried the c.4G>A (p.Ala2Thr) mutation, and their mother was strongly suspected to carry the mutated gene. Her sister developed diabetes around 40 years of age and required high doses of insulin, while the mother was diagnosed in her 20s and was managed with oral hypoglycemic agents; neither of them were obese. CONCLUSION: p.Ala2Thr mutation carriers often experience relatively later onset and normal BMI. Treatment regimens vary between individuals.

Novel Pathogenic De Novo INS p.T97P Variant Presenting With Severe Neonatal DKA.

Pathogenic INS gene mutations are causative for mutant INS-gene-induced diabetes of youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response.