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Severe congenital neutropenia (SCN) comprises a diverse range of rare hematological disorders characterized by recurrent, often life-threatening infections that manifest within the first months of life. Mutations in the ELANE gene are the most prevalent cause of SCN. While over 230 mutations in ELANE have been documented, including substitutions, frameshifts, nonsense mutations, and splice site alterations, the occurrence of deep intronic mutations has not been previously reported. Herein, we present the case of a young girl who exhibited recurrent fever, respiratory infections, skin abscesses, and gingivitis shortly after birth. Laboratory analysis revealed markedly diminished neutrophil levels alongside elevated monocyte and eosinophil counts. Bone marrow examination disclosed a halt in myelopoiesis maturation. ELANE gene full-length sequencing identified a novel de novo deep intron mutation in ELANE (c.598 + 79G > T), subsequently confirmed by Sanger sequencing. cDNA sequencing of the patient demonstrated aberrant gene splicing. Utilizing a mini-gene splicing assay for ELANE intronic variants, we identified a mutant ELANE allele (c.597 + 1_597 + 83ins) leading to the creation of a premature termination codon (p.Gly200ValfsTer40). Confocal microscopy revealed heightened expression of myeloperoxidase and neutrophil elastase in the patient, suggesting a potential role for the unfolded protein response in the pathogenesis of the deep intron ELANE mutation. In summary, our findings illustrate the first reported instance of de novo deep intron ELANE mutations associated with SCN, underscoring the importance of exploring deep intronic regions in SCN patients lacking identifiable disease-causing gene mutations.

Recent research in urothelial carcinoma (UC) has focused on coding mutations, leaving the significance of non-coding mutations unexplored. This study aims to evaluate non-coding DNA mutation frequencies compared to coding regions in normal urothelium and flat lesions, exploring their implications for tumor biology. Using targeted next-generation sequencing with UC-related gene panel, we analyzed non-coding and coding DNA mutation frequencies across 119 samples of flat urothelium encompassing various lesion types. Mutation patterns were examined based on the presence of associated flat or papillary tumors, and we investigated the correlation between mutation rates in target genes and genetic mutations within genomic regions. Intronic mutations (IMs) displayed variability across lesions, with normal urothelium (NU) exhibiting the highest frequency (43%) and urothelial carcinoma in situ (CIS) the lowest (9%). We observed similar sets of frequently mutated genes in both intronic and exonic regions, distinct from promoter region mutations. Although IMs paralleled exonic mutations in NU, reactive atypia, and atypia of unknown significance (AUS), they were less prevalent in dysplasia (DYS) and CIS. In contrast to CIS-associated AUS and DYS lesions, AUS-DYS lesions associated with papillary tumors exclusively exhibited recurrent intronic mutations involving FGFR3 and ERCC2 , aligning with mutation patterns seen in exonic regions. ERCC2 intronic mutations correlated with the mutation rates of the gene panel. Our findings suggest that intronic mutations significantly contribute to tumor heterogeneity in urothelial lesions and may potentially be linked to genomic instability, warranting further investigation.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss of function mutations in either TSC1 or TSC2. Genotype and phenotype analyses are conducted worldwide, but there have been few large-scale studies on Japanese patients, and there are still many unclear points. This study analyzed 283 Japanese patients with TSC (225 definite, 53 possible, and 5 genetic diagnoses). A total of 200 mutations (64 TSC1, 136 TSC2) were identified, of which 17 were mosaic mutations, 11 were large intragenic deletions, and four were splicing abnormalities due to deep intronic mutations. Several lesions and symptoms differed in prevalence and severity between TSC1 and TSC2 patients and were generally more severe in TSC2 patients. Moreover, TSC2 missense and in-frame mutations may attenuate skin and renal symptoms compared to other TSC2 mutations. Genetic testing revealed that approximately 20% of parents of a proband had mild TSC, which could have been missed. The patient demographics presented in this study revealed a high frequency of TSC1 patients and a low prevalence of epilepsy compared to global statistics. More patients with mild neuropsychiatric phenotypes were diagnosed in Japan, seemingly due to a higher utilization of brain imaging, and suggesting the possibility that a significant amount of mild TSC patients may not be correctly diagnosed worldwide.

Background Meier–Gorlin syndrome (MGORS) is a rare autosomal inherited form of primordial dwarfism. Pathogenic variants in 13 genes involved in DNA replication initiation have been identified in this disease, but homozygous intronic variants have never been reported. Additionally, whether growth hormone (GH) treatment can increase the height of children with MGORS is unclear. Methods The medical history data of a young girl were collected and reviewed. Whole-exome sequencing (WES) and bioinformatic analysis were performed to identify any variants and predict their pathogenicity. Minigene constructs were generated and transfected into HEK-293T cells for in vitro splicing assays. The literature was reviewed to explore the mutational spectrum and efficacy of GH treatment for this disease. Results A girl with microtia, hypoplastic patellae, and severe growth retardation carried a novel homozygous intronic variant (NM_030928.4: exon 3: c.352–30 A > C) in CDT1 . The variant was predicted to break a branch point and alter splicing, and the minigene assay confirmed abnormal splicing with exon 3 skipping. The patient was treated with GH for 5 years, with an increase in growth velocity from 4.0 cm/year to an average of 6.2 cm/year. A literature review revealed that the most common variant type and inheritance state were missense and compound heterozygous, respectively. Additionally, the vast majority of children with MGORS treated with GH had normal insulin-like growth factor 1 (IGF-1) levels, and half of them responded positively to GH therapy. Conclusions We reported a novel pathogenic homozygous intronic variant (c.352–30 A > C) of CDT1 in a girl with MGORS, and this mutation extended the genetic spectrum of the disease. GH therapy may be beneficial for height outcomes in children with MGORS with normal IGF-1 levels.

Background Fabry disease (FD, OMIM #301500) is an X-linked lysosomal disorder caused by the deficiency of α-galactosidase A (α-GalA), encoded by the GLA gene. Among more than 1100 reported GLA mutations, few were deep intronic mutations which have been linked to classic and cardiac variants of FD. Methods and results We report a novel hemizygous deep intronic GLA mutation (IVS4+1326C>T) in a 33-year-old Chinese man with a mild α-GalA deficiency phenotype involving isolated proteinuria and predominant globotriaosylceramide deposits in podocytes. IVS4+1326C>T, which appears to be the first deep intronic GLA mutation associated with renal variant of FD, was identified by Sanger sequencing the entire  GLA genomic DNA sequence of the patient’s peripheral mononuclear blood lymphocytes (PBMCs). Further sequencing of cDNA from PBMCs of the patient revealed a minor full-length GLA transcript accounting for about 25% of total GLA transcript, along with two major aberrantly spliced GLA transcripts encoding mutant forms of α-GalA with little enzyme activity characterized by in vitro α-GalA overexpression system in the HEK293T cells. Thus, the combined clinical phenotype, genetic analysis and functional studies verified the pathogenicity of IVS4+1326C>T. Conclusions The identification of IVS4+1326C>T establishes a link between deep intronic GLA mutation and the renal variant of FD, which extends the mutation spectrum in GLA gene and justifies further study of how IVS4+1326C>T and potentially other deep intronic GLA mutations contribute to Fabry podocytopathy through aberrant splicing. Future studies should also assess the true incidence of IVS4+1326C>T in patients with different variants of FD, which may improve early genetic diagnosis to allow timely treatment that can prevent disease progression and improve survival.

Aims Colony stimulating factor 1 receptor (CSF1R)‐related leukoencephalopathy is a rapidly progressing neurodegenerative disease caused by CSF1R gene mutations. This study aimed to identify and investigate the effect of a novel intronic mutation (c.1754‐3C>G) of CSF1R on splicing. Methods A novel intronic mutation was identified using whole‐exome sequencing. To investigate the impact of this mutation, we employed various bioinformatics tools to analyze the transcription of the CSF1R gene and the three‐dimensional structure of its encoded protein. Furthermore, reverse transcription polymerase chain reaction (RT‐PCR) was performed to validate the findings. Results A novel mutation (c.1754‐3C>G) in CSF1R was identified, which results in exon 13 skipping due to the disruption of the 3′ splice site consensus sequence NYAG/G. This exon skipping event was further validated in the peripheral blood of the mutation carrier through RT‐PCR and Sanger sequencing. Protein structure prediction indicated a disruption in the tyrosine kinase domain, with the truncated protein showing significant structural alterations. Conclusions Our findings underscore the importance of intronic mis‐splicing mutations in the diagnosis and management of CSF1R‐related leukoencephalopathy. A novel intronic mutation (c.1754‐3C>G) in CSF1R was identified using whole‐exome sequencing, that leads to exon 13 skipping due to the disruption of the 3′ splice site consensus sequence NYAG/G. Protein structure analysis indicated a disruption in the tyrosine kinase domain, with the truncated protein showing significant structural alterations.

A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel–Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.

BackgroundCharcot–Marie–Tooth (CMT) disease is a prevalent and heterogeneous peripheral neuropathy. Most patients affected with the axonal form of CMT (CMT2) do not harbor mutations in the approximately 90 known CMT-associated genes. We aimed to identify causative genes in two CMT2 pedigrees.MethodsNeurologic examination, laboratory tests and brain MRIs were performed. Genetic analysis included exome sequencing of four patients from the two pedigrees. The predicted effect of a deep intronic mutation on splicing was tested by regular and real-time PCR and sequencing.ResultsClinical data were consistent with CMT2 diagnosis. Inheritance patterns were autosomal recessive. Exome data of CMT2-101 did not include mutations in known CMT-associated genes. Sequence data, segregation analysis, bioinformatics analysis, evolutionary conservation, and information in the literature strongly implicated HADHA as the causative gene. An intronic variation positioned 23 nucleotides away from following intron/exon border in GDAP1 was ultimately identified as cause of CMT in CMT2-102. It was shown to affect splicing.ConclusionThe finding of a HADHA mutation as a cause of CMT is of interest because its encoded protein is a subunit of the mitochondrial trifunctional protein (MTP) complex, a mitochondrial enzyme involved in long chain fatty acid oxidation. Long chain fatty acid oxidation is an important source of energy for skeletal muscles. The mutation found in CMT2-102 is only the second intronic mutation reported in GDAP1. The mutation in the CMT2-102 pedigree was outside the canonical splice site sequences, emphasizing the importance of careful examination of available intronic sequences in exome sequence data.

Inherited optic neuropathies (ION) present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2′O-methyl-antisense oligonucleotides (AONs) with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (≃55%) using the cryptic acceptor splice sites targeting AON and moderate rescue (≃16%) using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ≃3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs.

Autosomal recessive mutations in genes required for cytotoxicity are causative of a life-threatening, early-onset hyperinflammatory syndrome termed familial hemophagocytic lymphohistiocytosis (FHL). Mutations in cause FHL type 3. encodes Munc13-4, a member of the Unc13 protein family which control SNARE complex formation and vesicle fusion. We have previously identified FHL3-associated mutations in the first intron of which control transcription from an alternative transcriptional start site. Using isoform specific antibodies, we demonstrate that this alternative Munc13-4 isoform with a unique N-terminus is preferentially expressed in human lymphocytes and platelets, as compared to the conventional isoform that was mostly expressed in monocytes and neutrophils. The distinct N-terminal of the two isoforms did not impact on Munc13-4 localization or trafficking to the immunological synapse of cytotoxic T cells. Moreover, ectopic expression of both isoforms efficiently restored exocytosis by FHL3 patient-derived Munc13-4 deficient T cells. Thus, we demonstrate that the conventional and alternative Munc13-4 isoforms have different expression pattern in hematopoietic cell subsets, but display similar localization and contribution to T cell exocytosis. The use of an alternative transcriptional starting site (TSS) in lymphocytes and platelets could be selected for increasing the overall levels of Munc13-4 expression for efficient secretory granule release.