Generation and characterization of induced pluripotent stem cells from a Parkinson's disease patient carrying the digenic LRRK2 p.G2019S and GBA1 p.N409S mutations.

peer reviewedWe describe an induced pluripotent stem cell (iPSC) line that was derived from fibroblasts obtained from a Parkinson's disease (PD) patient carrying the p.G2019S mutation in the LRRK2 gene and the p.N409S mutation in the GBA1 gene. iPSCs were generated via Sendai virus transduction of Yamanaka factors. The presence of GBA1 p.N409S and LRRK2 p.G2019S was confirmed by Sanger sequencing. The iPSCs express pluripotency markers, are capable of in vitro differentiation into the three germ layers and have a normal karyotype. The newly generated line will be used for in vitro PD modeling by investigating the role of each mutation in iPSC-derived dopaminergic neurons

Generation of induced pluripotent stem cell lines from a patient with Sotos syndrome carrying 5q35 microdeletion

Sotos syndrome (SoS) is a neurodevelopmental disorder caused by haploinsufficiency of the NSD1 gene located on chromosome 5 region q35.3. In order to understand the pathogenesis of Sotos syndrome and in view of future therapeutic approaches for its efficient treatment, we generated two human induced pluripotent stem cells (iPSCs) lines from one SoS patient carrying a 5q35 microdeletion. The established iPSCs expressed pluripotency markers, showing the capacity to differentiate into the three germ layers

<i>NSD1</i> Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint

An increasing amount of evidence indicates the critical role of the NSD1 gene in Sotos syndrome (SoS), a rare genetic disease, and in tumors. Molecular mechanisms affected by NSD1 mutations are largely uncharacterized. In order to assess the impact of NSD1 haploinsufficiency in the pathogenesis of SoS, we analyzed the gene expression profile of fibroblasts isolated from the skin samples of 15 SoS patients and of 5 healthy parents. We identified seven differentially expressed genes and five differentially expressed noncoding RNAs. The most upregulated mRNA was stratifin (SFN) (fold change, 3.9, Benjamini–Hochberg corrected p GSC) (fold change, 3.9, Benjamini–Hochberg corrected p p p NSD1 is involved in cell cycle regulation and that its mutation can induce the down-expression of genes involved in tumoral and neoplastic differentiation. The results contribute to defining the role of NSD1 in fibroblasts for the prevention, diagnosis and control of SoS

Identification of alternative transcripts of NSD1 gene in Sotos Syndrome patients and healthy subjects

NSD1 gene (Nuclear Receptor Binding SET Domain Protein 1) encodes a methyltransferase that plays an important role in embryonic development. NSD1 is implicated in the transcription and methylation of histone H3 at lysine 36 (H3-K36), but the molecular mechanisms involved in these processes remain largely unknown. Pathogenic variants of NSD1 gene lead to Sotos syndrome, and have also been detected in some type of cancers, such as acute myeloid leukemia. In this study we have investigated NSD1 mRNA expression in fibroblast cell lines obtained from 14 Sotos patients and from 8 healthy controls. In addition to the expected NSD1 canonical transcript (isoform 1), we identified two additional, not yet reported, short NSD1 mRNA isoforms: NSD1 Delta 5 Delta 7 (isoform 2) and NSD1 Delta 19-23 (isoform 3), both in healthy subjects and in Sotos patients. We also show that NSD1 mutations in patients can be associated with a decreased level of NSD1 mRNA, as expected. Moreover, one patient, bearing the NSD1 variant c.6010-10G &gt; A, expressed an additional shorter transcript derived from an aberrant splicing event. These results may provide a basis to elucidate the impact of different NSD1 pathogenic variants on the het-erogeneity of phenotype associated with Sotos syndrome

Molecular Analysis and Reclassification of NSD1 Gene Variants in a Cohort of Patients with Clinical Suspicion of Sotos Syndrome

Sotos syndrome is a rare genetic disorder caused by haploinsufficiency of the NSD1 (nuclear receptor binding SET domain containing protein 1) gene. No clinical diagnostic consensus criteria are published yet, and molecular analysis reduces the clinical diagnostic uncertainty. We screened 1530 unrelated patients enrolled from 2003 to 2021 at Galliera Hospital and Gaslini Institute in Genoa. NSD1 variants were identified in 292 patients including nine partial gene deletions, 13 microdeletions of the entire NSD1 gene, and 115 novel intragenic variants never previously described. Thirty-two variants of uncertain significance (VUS) out of 115 identified were re-classified. Twenty-five missense NSD1 VUS (25/32, 78.1%) changed class to likely pathogenic or likely benign, showing a highly significant shift in class (p NSD1, we identified variants in additional genes (NFIX, PTEN, EZH2, TCF20, BRWD3, PPP2R5D) in nine patients analyzed by the NGS custom panel. We describe the evolution of diagnostic techniques in our laboratory to ascertain molecular diagnosis, the identification of 115 new variants, and the re-classification of 25 VUS in NSD1. We underline the utility of sharing variant classification and the need to improve communication between the laboratory staff and the referring physician

The clinical and molecular spectrum of the KDM6B-related neurodevelopmental disorder

International audienceDe novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders