Hyperinsulinemic hypoglycemia (HI) is a rare feature in individuals with coloboma, heart defects, atresia choanae, retardation of growth and development, genital abnormalities, and ear abnormalities (CHARGE) syndrome, though its underlying mechanisms remain poorly understood. We report a Chilean female proband with genetically confirmed CHARGE syndrome caused by a pathogenic variant in the CHD7 gene, who presented with HI in the neonatal period. Initial hypoglycemia was detected on days 2-3 of life, followed by recurrent episodes prompting biochemical investigation. On day 21, HI was biochemically confirmed. Comprehensive hormonal evaluation, including cortisol and growth hormone testing, excluded deficiencies in these hormones as contributing factors. Genetic screening of 22 known HI-associated genes revealed no pathogenic variants, supporting the hypothesis that HI in this case is related to CHARGE syndrome rather than being a coincidental finding. The patient responded well to diazoxide treatment, which allowed for maintenance of normoglycemia, with gradual dose reduction as glucose management normalized. This case, along with 2 previously reported cases, suggests that HI can be an integral part of CHARGE syndrome. Further research is needed to understand the mechanisms connecting CHD7 variants and HI and to refine management strategies for affected individuals.

Context:Recent genetic discoveries in congenital hyperinsulinism (HI) and advances in sequencing technology suggest that the diagnostic yield may be improved by rescreening in people with genetically unsolved HI. Objective:To evaluate this hypothesis in a nationwide cohort of individuals with a historical diagnosis of HI of unknown genetic cause. Methods:Twenty-seven probands, representing 77% of the genetically unsolved HI cases in Finland, underwent rescreening which targeted the coding regions of 18 known HI genes, and 5 relevant non-coding regions. The median age of the cohort was 21 years (range, 4-44 years). Participants had previously undergone a median of 3 genetic tests (range, 1-4), all of which yielded negative (n = 17) or inconclusive (n = 10) results. Results:Genetic rescreening was informative in 22% (6 of 27) of cases. Definitive genetic diagnoses were established in 4 (15%) participants. These included the detection of non-coding variants in the ABCC8, HK1, and SLC16A1 genes, and a GCK mosaic variant (8% allele fraction). In 2 (7%) cases, rescreening revised genetic results but did not provide a definitive genetic diagnosis. Conclusion:In this Finnish cohort, rescreening with a comprehensive gene panel provided new or revised diagnoses in 22% of cases, informing on medical management and recurrence risk. These findings emphasize the importance of regularly updating genetic testing strategies and highlight the clinical value of re-evaluating the need for rescreening in genetically unexplained HI cases even following clinical remission.

AIM:Deep intronic variants can disrupt splicing and cause monogenic disease but are missed by routine genetic testing. This study assessed the contribution of deep intronic variants to Wolcott-Rallison syndrome (WRS), a recessive disorder characterized by early-onset diabetes and progressive multisystem disease caused by loss-of-function EIF2AK3 variants. METHODS:We investigated a cohort of 116 individuals referred to the Exeter Genomics Laboratory for genetic testing who had diabetes diagnosed at ≤2 years and at least one additional feature consistent with WRS: hepatic dysfunction, skeletal abnormalities or developmental delay. No genetic cause had been identified after testing all known early-onset diabetes genes. We screened genome-sequencing data for rare homozygous intronic EIF2AK3 variants. Candidate variants predicted to affect splicing by SpliceAI were assessed using a minigene exon-trapping assay. RESULTS:We identified two rare homozygous intronic EIF2AK3 variants in two siblings. Only one variant, c.1651-180G>T, was predicted to disrupt splicing in silico. The two children, born to consanguineous parents, were diagnosed with early-onset diabetes (diagnosed at 1 year and 21 weeks), hepatic dysfunction, skeletal abnormalities, developmental delay, thyroid dysfunction, hip dysplasia and gait abnormalities. The minigene assay showed that c.1651-180G>T creates a cryptic donor splice site within intron 9, resulting in inclusion of a 79-nucleotide pseudoexon, causing a frameshift and premature stop codon. Using this evidence, the variant was reclassified as likely pathogenic according to ACMG/ACGS guidelines. CONCLUSIONS:We report the first deep intronic EIF2AK3 variant causing WRS, highlighting the need to consider systematic intronic analysis in unresolved cases.

OBJECTIVES:To describe the birth prevalence and characterise the genotype, phenotype and management of children with congenital hyperinsulinism (CHI) in Western Australia (WA). METHODS:A population-based retrospective study (2005-2024) of children with CHI was conducted at the tertiary paediatric centre in WA. Children with transient perinatal stress-induced HI and insulinoma were excluded. Data collection included clinical presentation, investigations and management. RESULTS:The birth prevalence was 1 in 17,394 per 100,000 live births. Of 40 CHI cases, 65 % were neonatal presentations. 32.5 % were symptomatic with seizures in 15 % of presentations. An aetiology was identified in 80 % (n=32); 64.5 % (n=21) had identified pathogenic gene variants with K-ATP channel defects in 14 children [ABCC8 (n=11), KCNJ11 (n=3)], the remainder had syndromic HI (n=11). 80 % (n=32) responded to medical therapies; 78 % (n=25) had an identified etiology. A pathogenic variant was identified in six of the seven infants with CHI which resolved in six months. Of those children who were diazoxide unresponsive (n=10), two stabilised on octreotide while eight (20 %) required surgical management, which was curative in focal lesions (n=2). Secondary diabetes post pancreatectomy (n=6) occurred at a mean age of 5.25 years. The cohort had high continuous glucose monitoring uptake (92 %) for glucose monitoring following subsidy with nine commenced in the neonatal period. CONCLUSIONS:The prevalence of CHI in WA is higher than the reported European-ancestry population due to early genetic evaluation of newborns with CHI. Correlation between genotype and phenotype is consistent with literature and supports genetic investigation of infants with CHI to inform management.

BACKGROUND:Identifying novel genetic causes of diabetes in the first 6 months of life (neonatal diabetes) can highlight genes and pathways essential for pancreatic beta-cell development and function in humans. Our aim was to uncover genetic aetiologies of neonatal diabetes. METHODS:We performed genome sequencing in 38 probands diagnosed with neonatal diabetes without an identified genetic cause, and their unaffected parents. Genes with de novo coding variants in ≥2 probands were followed up. Replication was performed in a separate cohort of 288 genetically unresolved individuals diagnosed with neonatal diabetes. FINDINGS:The de novo ACTB (p.Ser348Leu) variant was identified in two unrelated individuals. Both had diabetes onset soon after birth (1 and 8 days), low birthweight (-3.22SD and -3.98SD), and extra-pancreatic features (hearing loss and developmental delay in one; intestinal atresia in the other). The same ACTB (p.Ser348Leu) variant was reported in seven individuals in the literature; one individual had confirmed neonatal diabetes and a further two had hyperglycaemia. Extra-pancreatic features were similar to our probands (hearing loss in 3/7; neurodevelopmental features in 4/7; gastrointestinal atresia in 6/7). In total, 5/9 individuals with the ACTB (p.Ser348Leu) variant had neonatal diabetes or hyperglycaemia. None of the 96 cases with other ACTB pathogenic variants in the Human Gene Mutation Database had diabetes. INTERPRETATION:The identification of 3 individuals (two in this report and one from the literature) with neonatal diabetes and a de novo ACTB p.(Ser348Leu) variant supports ACTB as a previously unrecognised neonatal diabetes aetiological gene, most likely through a variant-specific mechanism. FUNDING:Diabetes UK; EFSD/NNF; NIHR, Wellcome Trust.

Autoimmune diabetes presenting in infancy or with additional autoimmune disorders can be the result of highly penetrant variants in key immune homeostasis genes. An example of this is observed with biallelic pathogenic variants in IL2RA (CD25), which causes immunodeficiency 41 (IMD41). IL2RA encodes the α-chain of the interleukin-2 receptor, which helps regulate the growth and activity of T cells. The diabetes phenotype in IMD41 has not been systematically described. We sequenced IL2RA in 290 individuals with diabetes diagnosed <6 months and in 64 individuals diagnosed <10 years who had additional autoimmunity. We also reviewed all previously reported IMD41 cases. We identified five new unrelated individuals with biallelic pathogenic IL2RA variants. Four presented with diabetes in the first month of life with very low/absent C-peptide, and all were GAD antibody-positive. Of 17 previously reported cases, 9 had diabetes, yielding a total of 14 of 22 (64%) with early-onset diabetes. No relationship between variant location/type and diabetes development was observed. Autoimmune diabetes is therefore a common and early feature of IMD41. IL2RA should be included in genetic testing panels for neonatal and monogenic autoimmune diabetes. These findings highlight the important role of IL2RA in immune tolerance and β-cell protection. ARTICLE HIGHLIGHTS:We report five new cases of neonatal or early childhood-onset diabetes caused by biallelic pathogenic variants in IL2RA. Autoimmune diabetes was the presenting feature in most individuals and occurs in 64% of all reported cases of immunodeficiency 41. Patients typically presented in infancy with diabetic ketoacidosis, low C-peptide, and GAD antibody positivity, highlighting an autoimmune etiology. The diabetes phenotype was consistent across cases but not linked to variant type or location. IL2RA should be included in genetic testing for neonatal diabetes and considered in children with diabetes plus immune dysregulation.

Context:The coexistence of diabetes and hyperinsulinemic hypoglycemia (HI) within a family is rare. Activating MAFA variants have been described in 3 families with this dual phenotype and are associated with sex-dependent clinical patterns. Methods:We studied a family where members were affected by HI (n = 5), diabetes (n = 5), or both conditions (n = 1). Clinical, biochemical, and imaging data were collected. Genetic testing for variants in known monogenic diabetes and HI genes was performed in the proband, followed by cascade testing in available relatives. Pancreatic tissue was examined by immunohistochemistry and immunofluorescence. Results:A heterozygous, p.(Ser64Phe) MAFA variant was identified in the male proband, who had presented with diabetes at 28 years and developed HI due to insulinomatosis 6 years later. The variant was confirmed in 5 relatives, 4 with HI and 1 with diabetes (including obligate heterozygotes). Among the 5 presenting with HI, 3 were female, whereas 4 of 6 presenting with diabetes were male, consistent with reported sex-dependent patterns.Increased nuclear expression of the transcription factor MafA (MAFA) was detected by immunofluorescence in insulin-positive tumor regions compared to the adjacent islets, supporting a pathogenic role for the variant in beta-cell regulation. Conclusion:We describe the fourth family with a pathogenic MAFA variant and the second individual with the dual phenotype of diabetes and HI. Our findings highlight challenges in clinical management of this condition and underscore the need to consider MAFA in cases of adult-onset HI or those with an atypical course of diabetes especially when there is family history.

BACKGROUND:A substantial proportion of individuals with a well-defined monogenic disorder remain without a genetic diagnosis. Low-level mosaic pathogenic variants are recognised as an underappreciated cause of monogenic disease but are technically challenging to detect, particularly in organ-specific conditions when affected tissue is inaccessible. METHODS:We systematically investigated low-level mosaic variants in individuals with congenital hyperinsulinism (CHI: n = 1252) or neonatal diabetes (NDM: n = 312), two opposing pancreatic disorders of insulin secretion. We screened for established pathogenic variants with variant allele fraction (VAF) < 8% in dominant CHI (ABCC8, GCK, GLUD1, HK1) or dominant NDM (ABCC8, KCNJ11, INS) genes in targeted next-generation sequencing (tNGS) data using Mutect2. FINDINGS:This called 40 variants across the four genes in 39 individuals with CHI. No candidate variants were found in the NDM cohort. Orthogonal validation of 35 variants using TaqMan-based droplet digital PCR (ddPCR) confirmed 26/35 variants. The median VAF for confirmed variants was 3.6% (1.0-7.8%), while false positives (9/35) predominantly had a VAF <1% with some overlap in VAF with true positives. INTERPRETATION:This study shows that disease-causing low-level mosaic variants in dominant CHI genes can be detected in blood using tNGS but require orthogonal validation. These results provide a framework to improve diagnostic yield in organ-specific conditions where mosaic variants may represent an important missed cause of disease. FUNDING:This work was supported by a research grant from the University of Pennsylvania Orphan Disease Center in partnership with the Team CHIbra and Congenital Hyperinsulinism International [MDBR-23-020-CHI] and the Wellcome Trust [223187/Z/21/Z].

Schaaf-Yang syndrome and Prader-Willi syndrome are imprinting disorders that result from the disruption of paternally expressed genes within the 15q11-q13 region. Both conditions present with overlapping clinical features including developmental delay, hypotonia and endocrine abnormalities. Schaaf-Yang syndrome specifically results from heterozygous variants in the paternally expressed MAGEL2 gene. Because these variants are often de novo, determining that the variant is on the paternal allele is essential for a definitive diagnosis. Traditional methods, such as methylation-specific PCR, are labour-intensive, while it is challenging to phase variants separated by distances greater than the fragment length (~600 bp) using short-read sequencing. In this study, we used long-read Oxford Nanopore sequencing to perform trio-assisted phasing of a de novo MAGEL2, p.(Gln638Ter) variant identified in two unrelated probands referred for congenital hyperinsulinism genetic testing. Long reads spanning both the variant and informative parental heterozygous variants confirmed that the variant was on the paternal allele and was therefore pathogenic and causative of Schaaf-Yang syndrome and associated with hyperinsulinism in both individuals. Our findings demonstrate the clinical utility of long-read sequencing for enabling trio-assisted variant phasing in imprinting disorders, particularly when phenotypes are incomplete or overlapping. Our findings further highlight congenital hyperinsulinism as a rare but important feature associated with Schaaf-Yang syndrome.

Aims/hypothesis Accurate interpretation of loss-of-function (LOF) variants in MODY genes is essential for diagnosis but remains challenging, particularly for variants that are predicted to escape nonsense-mediated decay (NMD). We aimed to systematically evaluate the pathogenicity of LOF variants, stratified by NMD-triggering and NMD-escape status, across all known MODY genes. Methods We analysed ultra-rare LOF variants (minor allele frequency <1 in 10,000) in 5171 individuals of European ancestry with suspected MODY, compared with 155,501 population-based control individuals from UK Biobank. LOF variants in ABCC8, GCK, HNF1A, HNF4A, HNF1B, INS, KCNJ11, NEUROD1, PDX1 and RFX6 were classified as NMD-triggering or NMD-escape. We tested for gene-level enrichment in cases vs controls. For novel associations, we performed replication in additional MODY patients, assessed familial co-segregation, and undertook in silico protein modelling. Results LOF variants were significantly enriched in all MODY genes except ABCC8 and KCNJ11. Both NMD-triggering and NMD-escape variants were enriched in GCK, HNF1A and HNF4A, consistent with haploinsufficiency (all p<10(-3)). HNF1B and RFX6 showed enrichment only for NMD-triggering variants, while NEUROD1 and PDX1 were enriched only for NMD-escape variants. A novel finding was the significant enrichment of only NMD-escape LOF variants in INS (OR=181, p<10(-5)). Including replication in additional MODY patients, we identified eight families with 17 affected individuals carrying INS variants. These variants co-segregated with diabetes (logarithm of the odds score=3), included one de novo case, and were absent from >800,000 population control individuals. Individuals presented with diabetes at a median age of 19 years, had median BMI of 22.9 kg/m(2), were negative for islet autoantibodies, and had low type 1 diabetes genetic risk scores. Compared with INS missense MODY, diagnosis occurred approximately 10 years later in individuals with NMD-escape LOF variants. Protein modelling suggested that INS NMD-escape variants produce aberrant proinsulin molecules with unpaired B-chain cysteines, leading to milder misfolding. Conclusions/interpretation The pathogenicity of LOF variants in MODY genes depends on gene context and NMD status. Heterozygous NMD-escape LOF variants in INS are a novel cause of MODY. These findings provide systematic gene-level evidence to inform variant interpretation guidelines and improve the accuracy of MODY diagnosis in clinical practice.

Infants exposed to hypoglycaemia are at risk of neuroglycopenia and long-term neurodisability. Individuals with congenital hyperinsulinism provide a unique opportunity to gain insights into relevant biological pathways and outcomes. Such insights could transform clinical care for infants at risk of hypoglycaemia from a variety of genetic and environmental aetiologies.

Non-protein-coding genes are emerging as critical contributors to the etiology of rare diseases, providing key insights into human biology and uncovering novel disease mechanisms. We identified 7 individuals from 4 families with early-onset diabetes (diagnosed aged <5 years) and immune dysregulatory features caused by bi-allelic variants in RNU6ATAC. RNU6ATAC encodes a small nuclear RNA (snRNA) that acts as a catalytic component of the minor spliceosome, a protein-RNA complex that mediates the splicing of ∼700 genes containing U12/minor-type introns. Variant screening of the other 64 minor spliceosome genes in 276 infants with diabetes identified 12 unrelated individuals with bi-allelic disease-causing variants in RNU4ATAC. Bi-allelic pathogenic RNU4ATAC variants are known to cause a variable spectrum of clinical features, which until now did not include diabetes. Clinically, 12/19 RNU6ATAC/RNU4ATAC affected individuals had additional immune dysregulatory features, and 50% of individuals tested were islet-autoantibody positive, strongly supporting an autoimmune etiology for their diabetes. RNA sequencing (RNA-seq) in 3 individuals with bi-allelic RNU6ATAC variants showed a pattern of intron retention in U12-intron-containing genes similar to that seen in RNU4ATAC individuals (n = 3), supporting a shared disease mechanism. Analysis of affected individuals’ transcriptomic, methylation, and immune data revealed impaired B cell development and maturation. We conclude that bi-allelic RNU6ATAC variants cause a syndrome of early-onset autoimmune diabetes and immune dysregulation. We further show that infancy-onset diabetes is a feature of RNU4ATAC-opathy. Our work highlights the important role of two snRNAs critical to minor spliceosome function in immune system regulation, providing insights into the pathogenesis of autoimmune diabetes.

INTRODUCTION:Congenital hyperinsulinism (CHI) is a heterogeneous disorder of insulin dysregulation, leading to hypoglycemia. This study describes the clinical characteristics, genetics, and management of CHI in Argentina. METHODS:We retrospectively reviewed 70 probands diagnosed with CHI (2008-2021) at multiple centres across Argentina. Clinical, biochemical, imaging, and treatment data were analyzed. Genetic testing was performed in 49 probands using Sanger and targeted next-generation sequencing of CHI-related genes. RESULTS:Transient CHI was identified in 23/70 (33%) probands, with a median duration of 2 months. Risk factors for perinatal stress-induced hyperinsulinism (PSHI) were present in 85% of transient cases. Persistent CHI was diagnosed in 44/70 (63%) individuals, of whom 31 responded to diazoxide. Late-onset CHI (diagnosed >3 years) was identified in 3 children. A pathogenic variant was detected in 19/49 (39%) probands, all had persistent CHI. ABCC8 variants were most common accounting for 68% (13/19) of diagnoses. Imaging in 17 cases revealed focal disease in 8, diffuse disease in 8, and atypical disease in 1 individual. Seven individuals with focal disease underwent lesionectomy, which was curative in 5 (71%). Three children with diffuse disease required near-total pancreatectomy, with one developing postoperative diabetes. CONCLUSIONS:This study provides the largest CHI cohort reported from South America and highlights the clinical and genetic heterogeneity of the condition. Transient CHI was often associated with PSHI risk factors, while persistent CHI was predominantly linked to K-ATP channel variants. The findings underscore the importance of genetics and imaging for CHI management and emphasize the need for increased access to molecular diagnostics.

Aims/hypothesis Accurate interpretation of loss-of-function (LOF) variants in MODY genes is essential for diagnosis but remains challenging, particularly for variants predicted to escape nonsense-mediated decay (NMD). We aimed to systematically evaluate the pathogenicity of LOF variants, stratified by NMD-triggering and NMD-escape status, across all known MODY genes. Methods We analysed ultra-rare LOF variants (minor allele frequency <1 in 10,000) in 5171 individuals of European ancestry with suspected MODY, compared with 155,501 population-based controls from UK Biobank. LOF variants in ABCC8 , GCK , HNF1A , HNF4A , HNF1B , INS , KCNJ11 , NEUROD1 , PDX1 , and RFX6 were classified as NMD-triggering or NMD-escape. We tested for gene-level enrichment in cases versus controls. For novel associations, we performed replication in additional MODY cases, assessed familial co-segregation, and undertook in-silico protein modelling. Results LOF variants were significantly enriched in all MODY genes except ABCC8 and KCNJ11 . Both NMD-triggering and NMD-escape variants were enriched in GCK , HNF1A , and HNF4A , consistent with haploinsufficiency (all P <10-3). HNF1B and RFX6 showed enrichment only for NMD-triggering variants, while NEUROD1 and PDX1 were enriched for only NMD-escape variants. A novel finding was significant enrichment of only NMD-escape LOF variants in INS (OR = 181, P < 10-5). Including replication, we identified eight families with 17 affected individuals carrying INS variants. These variants co-segregated with diabetes (LOD score = 3), included one de novo case, and were absent from >800,000 population controls. Individuals presented with diabetes at a median age of 19 years, had median BMI of 22.9 kg/m2, were negative for islet autoantibodies, and had low type 1 diabetes genetic risk scores. Compared with INS missense MODY, diagnosis occurred ~10 years later. Protein modelling suggested that INS NMD-escape variants produce aberrant proinsulin molecules with unpaired B chain cysteines, leading to milder misfolding. Conclusions/interpretation The pathogenicity of LOF variants in MODY genes depends on gene context and NMD status. Heterozygous NMD-escape LOF variants in INS are a novel cause of MODY. These findings provide systematic gene-level evidence to inform variant interpretation guidelines and improve the accuracy of MODY diagnosis in clinical practice. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement European Foundation for the Study of Diabetes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: North Wales Research Ethics Committee (REC reference: 17/WA/0327) gave ethical approval for this work. The UK Biobank Research Ethics Committee (REC reference: 11/NW/0382) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes UK Biobank data is available at https://www.ukbiobank.ac.uk/enable-your-research and is accessible through application. GnomAD data is freely available to all at https://gnomad.broadinstitute.org/. The MODY cohort data is not publicly available for ethical and patient confidentiality related reasons but is available upon reasonable request to the corresponding author.