RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation, causing a distinct Mendelian disorder.

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present clinical, genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively acting germline alleles p.Arg180Gly and p.Gly183Arg, which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate phosphatidylinositol 3-phosphate (PI3P) and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts show accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

SNUPN deficiency causes a recessive muscular dystrophy due to RNA mis-splicing and ECM dysregulation.

SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.

A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing.

Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA-seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA-seq, we delineated a deep intronic TAPT1 mutation (c.1237-52 G>A) that segregated with the disease. Using SI-NET-seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre-mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein-null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease-relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non-coding variants, as well as in illuminating the molecular mechanisms of human diseases.

Bioenergetic analysis of aged-phenotype skin in a rare syndromic cutis laxa.

BACKGROUND: Skin aging is an inevitable phenomenon characterized by wrinkled skin and loss of elasticity. To date, several studies have been performed on skin aging to discover the underlying mechanisms and improve efficient preventive strategies and anti-aging therapeutics. AIMS: Here, we aimed to investigate the modifications of oxidative phosphorylation and glycolysis which are the critical determinants of aging in aged-phenotype skin. METHODS: Due to the complexity of the skin aging process, we performed bioenergetic measurements on aged-phenotype fibroblasts from an inherited cutis laxa syndrome which remarkably presents clinical features of normal aged skin. Bioenergetic analysis was performed on cutis laxa samples (n = 3) and healthy samples (n = 3) using Seahorse XFe24 Analyzer. We also compared the sensitivity of cultured aged-phenotype fibroblasts to normal cells in glucose withdrawal. RESULTS: Our results show a significant increase in oxidative phosphorylation parameters but not glycolysis in the patient fibroblast cells implying increased energy demand and preferential dependence on mitochondrial respiration in those cells. Interestingly, we found the patient cells demonstrate hypersensitivity to glucose starvation, supporting their enhanced energy consumption. CONCLUSIONS: In summary, our work suggested increased energy demand and higher oxidative phosphorylation in aged-phenotype cells which can be considered in anti-skin aging therapeutic design.

miR-324-3p and miR-508-5p expression levels could serve as potential diagnostic and multidrug-resistant biomarkers in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the most frequent hematological malignancies in children, representing approximately 25 % of all pediatric cancers. Despite striking advances in ALL treatments, a small population of patients does not still respond to chemotherapy, raising the number of deaths in children. ABC transporters are one of the major causes of multidrug resistance (MDR) in cancers and overexpression of ABCA3 is directly associated with increased chemo-resistance in pediatric ALL. Here, we aimed to identify the microRNAs (miRNAs) which may regulate the expression of ABCA3 in childhood ALL. Bone marrow samples from a total of 50 ALLs and 59 controls were collected and after in silico and literature search, miR-324-3p and miR-508-5p were nominated from a list of putative miRNAs targeting ABCA3. Our qPCR analysis showed a low expression profile of selected miRNAs in pediatric ALL patients compared with non-cancer controls. Furthermore, we found that both miR-324-3p and miR-508-5p were significantly differentially expressed between patients with positive and negative minimal residual disease (MRD + vs MRD-) after one year of chemotherapy while only miR-508-5p was underexpressed in relapsed ALL patients. Additionally, a negative correlation was identified between the expression of these two miRNAs and ABCA3, supporting the regulatory effect of them on drug resistance through interacting with ABCA3. Overall, we suggested miR-324-3p and miR-508-5p as potential diagnostic and drug-resistant biomarkers in pediatric ALL. Moreover, our findings presented miR-508-5p to behave as a promising relapsed indicator in childhood ALL which can be applied in the development of novel therapeutic strategies.