The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.

Objective: A significant proportion of individuals with suspected genetic developmental and epileptic encephalopathies (DEEs) remain unsolved following whole genome sequencing (WGS). Here we describe biallelic RNU2‐2 variants causing a recently reported, severe, recessive DEE. Methods: We screened individuals who have received WGS analyses at the Genomic Medicine Centre Karolinska for Rare Diseases for biallelic RNU2‐2 variants. Deep phenotyping was performed through reviewing entire medical histories and phenotypic traits were transcribed to their corresponding Human Phenotype Ontology (HPO) term. HPO terms were used to generate pairwise phenotypic similarity scores and assess for significantly shared phenotype enrichment in the RNU2‐2 sub‐cohort. RNA sequencing analyses were performed in fibroblast and blood tissues to compare splicing events between RNU2‐2 individuals and two independent control groups. Results: We identified 14 individuals from nine families with 12 ultra‐rare biallelic RNU2‐2 variants clustering in the conserved 5′ domains. Genotype data from 13 of 14 individuals has been reported previously as part of a larger cohort. All individuals presented with a highly concordant, severe DEE, characterized by severe to profound intellectual disability, inability to walk or communicate, hyperkinesia, and refractory seizures. Infantile spasms and tonic seizures were the predominant seizure types and a Lennox–Gastaut syndrome‐like phenotype was common. These individuals had a significantly similar phenotypic signature when compared with 703 individuals with complex pediatric epilepsies (two‐sided Monte Carlo permutation test, p = .005). RNA sequencing analyses showed aberrant splicing, with the most pronounced effects in fibroblast tissues in mutually exclusive exon and alternate 3′ splice‐site events, which were not detectable in blood. Significance: We present deep phenotyping data and transcriptomic analyses that provide support for rare, 5′ clustering biallelic RNU2‐2 variants causing this novel, severe DEE. We propose an RNA sequencing methodology on fibroblast tissue for future validation of RNU2‐2 variants.

Sweden has one neonatal screening laboratory and two centers conducting diagnostic workup for inborn errors of metabolism (IEM). Next-generation sequencing (NGS) has been gradually introduced as a confirmatory diagnostic test in the Swedish newborn screening program. Here, we describe the use of NGS in the diagnostic workup of IEM in screening-detected babies in Sweden between 2015 and 2023. During this period, 1,023,344 newborn children were screened, and 81 of 290 IEM cases were genetically confirmed using NGS. Planned improvements to the program are to perform genetic validation directly on the initial dried blood spot (DBS). As whole-genome sequencing (WGS) is superior in detecting causative genetic variants compared to Sanger sequencing, targeted NGS, and whole-exome sequencing (WES), it will likely become the method of choice more broadly in the future. A strong focus is to consolidate the nationally coordinated DBS newborn screening program, with all its individual components, including screening, targeted diagnostics, individualized treatment, and follow-up. This challenges the current regionalized organization of Swedish healthcare, which hinders close national collaboration between experts and sharing of data, as well as equal access to advanced treatments for identified patients, regardless of their place of birth.