A novel stop-loss mutation in NKX2-2 gene as a cause of neonatal diabetes mellitus: molecular characterization and structural analysis.

AIM: To identify the genetic etiology of neonatal diabetes in an infant and to elucidate the molecular mechanism of the identified mutation underlying the pathogenesis. METHODS: Genetic analysis was carried out by sequencing of known etiological genes associated with NDM. Molecular characterization was performed by constructing a identified mutation in NKX2-2 gene and  functional aspects was tested using transactivation, protein expression, DNA binding, nuclear localization assays. Structural analysis was performed by modeling the NKX2-2 protein structure. RESULTS: A novel homozygous frameshift mutation  c.772delC, p.Q258SFs*59 in the NKX2-2 gene was identified in a patient with neonatal diabetes. Functional studies revealed that this mutation resulted in an elongated protein sequence, affecting DNA binding activity and transcriptional function. Structural analysis suggested alterations in the protein's tertiary structure, likely contributing to its dysfunction. CONCLUSION: This study presents the first report of a stop-loss mutation in the NKX2-2 gene associated with NDM. Our findings emphasize the importance of functional and structural characterization to understand the biological consequences of such mutations. This comprehensive analysis provides insights into the molecular mechanisms underlying NDM and its clinical phenotype, which may aid in better diagnosis and management of patients with similar variants in the future.

Molecular characterization and re-interpretation of HNF1A variants identified in Indian MODY subjects towards precision medicine.

Background: HNF1A is an essential component of the transcription factor network that controls pancreatic ß-cell differentiation, maintenance, and glucose stimulated insulin secretion (GSIS). A continuum of protein malfunction is caused by variations in the HNF1A gene, from severe loss-of-function (LOF) variants that cause the highly penetrant Maturity Onset Diabetes of the Young (MODY) to milder LOF variants that are far less penetrant but impart a population-wide risk of type 2 diabetes that is up to five times higher. Before classifying and reporting the discovered variations as relevant in clinical diagnosis, a critical review is required. Functional investigations offer substantial support for classifying a variant as pathogenic, or otherwise as advised by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) ACMG/AMP criteria for variant interpretation. Objective: To determine the molecular basis for the variations in the HNF1A gene found in patients with monogenic diabetes in India. Methods: We performed functional protein analyses such as transactivation, protein expression, DNA binding, nuclear localization, and glucose stimulated insulin secretion (GSIS) assay, along with structural prediction analysis for 14 HNF1A variants found in 20 patients with monogenic diabetes. Results: Of the 14 variants, 4 (28.6%) were interpreted as pathogenic, 6 (42.8%) as likely pathogenic, 3 (21.4%) as variants of uncertain significance, and 1 (7.14%) as benign. Patients harboring the pathogenic/likely pathogenic variants were able to successfully switch from insulin to sulfonylureas (SU) making these variants clinically actionable. Conclusion: Our findings are the first to show the need of using additive scores during molecular characterization for accurate pathogenicity evaluations of HNF1A variants in precision medicine.

Human gain-of-function variants in HNF1A confer protection from diabetes but independently increase hepatic secretion of atherogenic lipoproteins.

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9. In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins.

Genotype-phenotype correlation of KATP channel gene defects causing permanent neonatal diabetes in Indian patients.

BACKGROUND: There are very few reports pertaining to Indian patients with neonatal diabetes mellitus (NDM). Activating or gain of function mutations of KATP channel genes namely KCNJ11 and ABCC8 are most predominant cause of permanent neonatal diabetes mellitus (PNDM). OBJECTIVES: To identify the genotype-phenotype correlation of KATP channel gene defects in a large series of (n = 181) Indian PNDM patients. METHODS: Direct sequencing of all exons of KCNJ11 and ABCC8 genes in all 181 patients with PNDM were performed. Clinical and biochemical data were collected. RESULTS: We have identified the molecular basis of KATP -NDM in 39 out of 181 patients (22%). Of these, 20 had KCNJ11 mutations and 19 had ABCC8 mutations, thus comprising 51% of KCNJ11 and 49% of ABCC8. There were four novel mutations (D1128Tfs*16, Y1287C, S1422T, and H1537R) in ABCC8 gene. Three patients with KCNJ11 mutations had developmental delay with DEND syndrome. In patients with ABCC8 mutations developmental delay was seen in seven out of 19 (36.8%). Of this, three patients (15.7%) had DEND phenotype and four (21%) had iDEND. Of the 39 patients, 33 (84%) patients were shifted to sulfonylurea therapy (glibenclamide). Of this, 19(57.5%) patients harbored KCNJ11 mutations and 14(42.1%) ABCC8 mutations. CONCLUSIONS: This is the first largest study in NDM patients in India demonstrating the importance of KATP channel gene mutation screening in PNDM and efficacy of glibenclamide for Indian patients with KATP -PNDM. The success rate of transfer is more in patients with KCNJ11 mutations compared with those with ABCC8 mutations.

Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children.

BACKGROUND: Gain-of-function of ATP-sensitive K+ (KATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in KATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. METHODS: Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known KATP inhibitors in intact cells were determined by 86 Rb efflux assays. RESULTS: Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP4- inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the KATP channel inhibitors glibenclamide and carbamazepine. CONCLUSIONS: The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect KATP channel function to cause the disease. The reduced ATP4- sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.