Elisa De Franco

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.

A substantial proportion of individuals with a well-defined monogenic disorder remain without a genetic diagnosis. Low-level mosaic pathogenic variants are increasingly recognised as an underappreciated cause of monogenic disease but are technically challenging to detect, particularly in organ-specific conditions when affected tissue is inaccessible. We systematically investigated low-level mosaic variants in individuals with congenital hyperinsulinism (CHI: n=1,252) or neonatal diabetes (NDM: n=312), two opposing pancreatic disorders of insulin secretion. We screened for established pathogenic variants with variant allele fraction (VAF) ABCC8 , GCK , GLUD1 , HK1 ) or dominant NDM ( ABCC8 , KCNJ11 , INS ) genes in targeted next generation sequencing (tNGS) data using Mutect2. 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% (1.1–7.8%), while false positives (9/35) predominantly had a VAF <1% with some overlap in VAF with true positives. 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.

Objectives: Monogenic diabetes (MD) is a group of diabetes subtypes caused by defects in single genes. We report phenotypes and genotypes of MD among Sudanese children. Methods: Referred patients (from birth to 18 years of age) with diabetes and a clinical diagnosis of MD to Gaafar Ibnauf Pediatric Tertiary Hospital or the Sudan Childhood Diabetes Center between January 2006 and April 2023 were included. Patients were divided into two groups based on onset of diabetes before six months of age (Group 1, or neonatal diabetes mellitus) or after (Group 2, or non-neonatal diabetes mellitus). Genetic testing was performed for 87 patients at the Exeter Genomics laboratory and for one patient at the University of Cambridge, Metabolic Research Laboratories, UK. Results: Out of 88 patients, 50 were from Group 1 and 38 from Group 2. We reported consanguinity in 63.6 % of the cohort and identified disease-causing variants for 18 genes in 43.2 % (Group 1) and 37.5 % (Group 2) of patients from the total cohort. The commonest causes in Group 1 and Group 2 were pathogenic variants in the EIF2AK3 and WFS1 genes, respectively. Pathogenic variants in recently reported novel genes ZNF808 , NARS2 , and FICD were detected in 8.5 %, 4.2%, and 1.4 % of patients, respectively. Patients with a disrupted WFS1 gene were found to have deafness (92.8 %) and optic atrophy (64 %). While skeletal deformities and liver disease were both seen in 28.6 % of patients with pathogenic variants in the ElF2AK3 gene. Hepatomegaly and hypophosphatemic rickets were uniformly seen in patients with pathogenic variants in the SLC2A2 gene. Generalized subcutaneous tissue loss and acanthosis nigricans were main features in AGPAT2 and INSR variants, respectively. Conclusions: Characterization of MD in Sudan showed a predominance of syndromic forms. Genetic studies conducted on consanguineous populations may raise higher probabilities in identifying rare genes.

Pathogenic variants in non-coding genes are emerging as critical contributors to human rare diseases. We identified 19 individuals with early-onset diabetes (diagnosed RNU6ATAC (n=7) or in RNU4ATAC (n=12). Both genes encode non-coding components of the minor spliceosome, a protein-RNA complex mediating splicing of ∼700 genes containing U12/minor-type introns. RNA-seq of whole blood from six patients showed aberrant splicing of minor intron-containing genes in individuals with RNU6ATAC (n=3) or RNU4ATAC (n=3) variants. 50% of patients tested were islet-autoantibody positive, and 12/19 had additional immune features of immune dysregulation. Analysis of patients’ transcriptomic, methylation and immune data revealed impaired B cell development and maturation. Biallelic disease-causing variants in RNU6ATAC and RNU4ATAC therefore cause syndromic early-onset autoimmune diabetes and immune dysregulation, highlighting the critical role of the minor spliceosome as a master regulator of the human immune system.

Background: Recessive loss‐of‐function NARS2 variants causing the multi‐system disorder Combined oxidative phosphorylation deficiency 24 (COXPD24) have recently been reported in 3 individuals with diabetes diagnosed between 3 days and 14 months of age. In this study, we investigate the presence of NARS2 variants in a large cohort of individuals with early‐onset diabetes. Methods: We used genome and targeted next‐generation sequencing to screen for rare, coding biallelic NARS2 variants in a cohort of 397 individuals diagnosed with diabetes NARS2 (4 individuals with the p.(Phe216Leu) variant, 3 with p.(Thr180Asn) and one with p.(Val440Leu)). All 8 individuals were diagnosed with insulin‐dependent diabetes before 6 months of age (neonatal diabetes, NDM) with the median age at diagnosis being 4 weeks (range: 1 to 20 weeks). 7/8 probands had low birthweight (median Z ‐score: −2.43, range: −4.17 to 0.86). Neurological features were common, with epilepsy and developmental delay each identified in 7/8 and 6/8 participants, respectively. Conclusion: Taken together with previously published cases, this study shows that NDM is an important feature of COXPD‐24 and highlights a critical role for NARS2 in the insulin‐secreting pancreatic β‐cell.