Control Lymphoblastoid Cell Lines Research Articles

Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

Somatic and Germline Cohesin Genes Alterations in Pediatric Acute Lymphoblastic Leukemia

Leukemia is a complex disease and the molecular mechanisms of malignant transformation are not fully understood yet. Cohesins are essential proteins, forming a complex which plays critical roles in various cellular processes, not only by canonical DNA binding and chromosome segregation but also in non-canonical regulation of gene expression in both proliferating and post-mitotic cells. Mutations or gene expression alterations of genes encoding the components of the cohesins' complex may affect chromosomal stability and potentially contribute to the pathogenesis of leukemia. Somatic cohesins mutations have been already reported to occur in myeloproliferative disorders and pediatric Acute Lymphoblastic Leukemia (ALL), while on the other hand, germline mutations are causative of Cohesinopathies such as Cornelia de Lange Syndrome (CdLS). Our previous first report of a patient affected by both CdLS and ALL suggested a potential involvement of cohesin germline mutations in the pathogenesis of ALL. Moreover, we demonstrated that germline mutations in STAG1 gene could play a crucial role in predisposition to pediatric hematological malignancies, including ALL. These findings suggest that both somatic and germline alterations of cohesin genes may be associated with the development of ALL. This study aims to understand the molecular mechanisms affected by Cohesin genes alterations, that could potentially pave the way for the development of targeted therapies and improved treatment strategies for leukemia patients. By employing a DNA-targeted next-generation sequencing (NGS) screening of a cohort of 120 consecutive pediatric ALL cases (enrolled in the AIEOP-BFM protocol at various Italian centers), we discovered a total of 11 germline cohesin mutations, further categorized as follows: 6 out of 11 were missense, 4 out of 11 were located in the 3' or 5' untranslated regions (UTR) and 1 out of 11 was a splice-acceptor variant. To investigate the functional implications of selected germline mutations in STAG1 gene, we took advantage of the CRISPR/Cas9 technique to generate in vitro models of two Lymphoblastoid Cell Lines (LCLs) harboring specific variants already found in two patients of our cohort. Additionally, we established control LCLs from healthy donors' samples using the same CRISPR/Cas9 approach. Subsequently, we performed RNA sequencing (RNAseq) analysis on these cell lines, and our preliminary findings revealed a total of 619 differentially expressed genes (DEGs) compared to the control group. Many of them are involved in critical intracellular pathways, like ribosomal transcription, suggesting potential disruptions of essential cellular processes caused by germline STAG1 mutations. Moreover, we have successfully developed an advanced computational analysis pipeline with the primary objective of detecting fusion transcripts from RNAseq data. This pipeline was applied to a dataset of 529 consecutive diagnose and 95 relapse samples of pediatric B/T-ALL patients and in five B-ALL cases a fusion transcript involving the STAG1 (n=1), STAG2 (n=3), and NIPBL (n=1) genes was identified. The significance of these findings was further reinforced by additional cases from both pediatric and adult cohorts through an international collaborative effort and five more fusion transcripts of STAG2 (n=2) and NIPBL (n=3) were uncovered. Investigation to clarify the underlying functional consequences of these fusion genes in pediatric ALL are ongoing. Collectively, these observations endeavor to elucidate the role of germline and somatic cohesin events in pediatric ALL. In particular, the investigation seeks to unveil the non-canonical functions of these proteins, thus paving the way for the identification and characterization of a novel and distinct subcategory of leukemia within the pediatric population.

Divergent Transcriptomic Effects of Allopregnanolone in Postpartum Depression.

Brexanolone, a formulation of the neurosteroid allopregnanolone (ALLO), is approved for treating postpartum depression (PPD) and is being investigated for therapeutic efficacy across numerous neuropsychiatric disorders. Given ALLO's beneficial effects on mood in women with PPD compared to healthy control women, we sought to characterize and compare the cellular response to ALLO in women with (n = 9) or without (n = 10, i.e., Controls) past PPD, utilizing our previously established patient-derived lymphoblastoid cell lines (LCLs). To mimic in vivo PPD ALLO-treatment, LCLs were exposed to ALLO or DMSO vehicle for 60 h and RNA-sequenced to detect differentially expressed genes (DEGs, pnominal < 0.05). Between ALLO-treated Control and PPD LCLs, 269 DEGs were identified, including Glutamate Decarboxylase 1 (GAD1), which was decreased 2-fold in PPD. Network analysis of PPD:ALLO DEGs revealed enriched terms related to synaptic activity and cholesterol biosynthesis. Within-diagnosis analyses (i.e., DMSO vs. ALLO) detected 265 ALLO-induced DEGs in Control LCLs compared to only 98 within PPD LCLs, with just 11 DEGs overlapping. Likewise, the gene ontologies underlying ALLO-induced DEGs in PPD and Control LCLs were divergent. These data suggest that ALLO may activate unique and opposing molecular pathways in women with PPD, which may be tied to its antidepressant mechanism.

Integrated microRNA-mRNA Expression Profiling Identifies Novel Targets and Networks Associated with Autism.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, with mutations in hundreds of genes contributing to its risk. Herein, we studied lymphoblastoid cell lines (LCLs) from children diagnosed with autistic disorder (n = 10) and controls (n = 7) using RNA and miRNA sequencing profiles. The sequencing analysis identified 1700 genes and 102 miRNAs differentially expressed between the ASD and control LCLs (p ≤ 0.05). The top upregulated genes were GABRA4, AUTS2, and IL27, and the top upregulated miRNAs were hsa-miR-6813-3p, hsa-miR-221-5p, and hsa-miR-21-5p. The RT-qPCR analysis confirmed the sequencing results for randomly selected candidates: AUTS2, FMR1, PTEN, hsa-miR-15a-5p, hsa-miR-92a-3p, and hsa-miR-125b-5p. The functional enrichment analysis showed pathways involved in ASD control proliferation of neuronal cells, cell death of immune cells, epilepsy or neurodevelopmental disorders, WNT and PTEN signaling, apoptosis, and cancer. The integration of mRNA and miRNA sequencing profiles by miRWalk2.0 identified correlated changes in miRNAs and their targets’ expression. The integration analysis found significantly dysregulated miRNA–gene pairs in ASD. Overall, these findings suggest that mRNA and miRNA expression profiles in ASD are greatly altered in LCLs and reveal numerous miRNA–gene interactions that regulate critical pathways involved in the proliferation of neuronal cells, cell death of immune cells, and neuronal development.

Potential Role of STAG1 Mutations in Genetic Predisposition to Childhood Hemato-Oncological Diseases

Background. Genetic predisposition has been recently assessed in about 5-10% of pediatric tumors, including leukemias. Among genetic syndromes associated with an increased risk of developing leukemia, a possible link has been proposed for Cohesinopathies, like Cornelia de Lange Syndrome (CdLS), caused by mutations in Cohesin family genes. Somatic mutation in these genes, known for their fundamental role in cell cycle and DNA repair mechanisms, are described in myeloid malignancies (10-20% of AML, 50% of DS-AMKL, 5-15% of MDS and 10% of MPN) and solid tumours. We previously described the first germline variant of NIPBL in a CdL case with acute lymphoblastic leukemia (ALL). The identification of germline variants can provide new insights in the pathogenesis of hemato-oncological diseases.Objectives. This study aims to define the association of germline variants of the Cohesin family genes with predisposition to pediatric hemato-oncological diseases, including ALL. Functional assays have been performed on a STAG1 unique variant.Methods. We set up an NGS capture-base DNA panel, including 40 genes associated with 8 pathways associated to ALL predisposition, including Cohesin genes. Patients' bone marrow (BM) diagnostic samples have been analyzed together with a germline tissue (BM at remission or buccal brush). Bioinformatic analysis has been carried out by Sophia DDM software (Variant Fraction >5%; Coverage at least 500X; Variant Allele Frequency 1%) and variants were annotated as certainly pathogenic, potentially pathogenic or VUS.A Lymphoblastoid Cell Line (LCL) was obtained from PB of a MDS patient, then used for functional studies.Genomic stability and X-ray induced DNA-damage repair were evaluated in the STAG1-mutated LCL by Sister Chromatids Exchange (SCE) assay, cell cycle analysis (Propidium Iodide) and pH2AX staining.Results. Overall, 117 consecutive pediatric ALL patients and sporadic cases with familial recurrency of cancer or other hematological disorders have been analyzed through the NGS panel. A total of 225 mutations were identified in ALL, distributed as it follows: 77 variants in Transcription factors, 68 in Signaling-associated genes, 56 variants in RAS pathway, 44 in chromatin modifier genes, 38 variants in B-cell differentiation genes, 30 in DNA repair genes and 16 variants in genes associated to predisposition. Overall, 12/117 unique variants (5%) were found in Cohesins family genes; 11/12 were germline, while a single somatic variant was found in STAG2 in the same position as a germline variant. In details, 3 mutations were found in BRD2 gene while 2 variants were identified in STAG1, STAG2 and NIPBL genes, respectively. Only 1 variant was detected in SMC1A, SMC3 and BRD2. No mutations were detected in HDAC8 and RAD21 genes.Interestingly, the Arg1167Gln variant in STAG1 is located in a highly conserved region involved in solid tumors. A STAG1 variant (Arg1187Gln) was identified in a pediatric MDS patient, which was then further characterized in the LCL. First, we evaluated the effects on chromosomal stability, and we observed a higher number of abnormal chromatids exchanges in mutated LCL (STAG1 patient mean = 4.8 exchanges/metaphase compared to four control LCLs mean = 3.05; p <0.0001), as shown in the attached figure. Differences in cell cycle phases between STAG1-mutated LCL and controls, even after X-ray irradiation (3Gy - 6Gy), are not significant. Mutated cells showed a significantly lower capability to repair DNA after an ionizing radiation. γH2AX phosphorylation status of STAG1 mutated LCL appears higher also in basal condition (T0: 1.7X, p <0.01; T24: 2.2X, p <0.0001; T48: 2.4X, p <0.0001; MFI STAG1 over MFI control LCLs) and it remains at higher levels at 48 hours after the irradiation (T48: 3.7X, p<0.001 [3Gy]; 4.1X, p<0.0001 [6Gy]; MFI STAG1 over MFI control LCLs). These data are confirmed by the ratio of the STAG1-mutated LCL median MFI over controls (T48/T0: 3.42 vs 0.92, p <0.0001 [3Gy]; T48/T0: 2.33 vs 0.64, p <0.01 [6Gy]).Conclusion. In pediatric hematological diseases, NGS screening showed several variants among Cohesin genes, with either a potentially pathogenic or unknown significance.Our study confirms a considerable effect of a STAG1 germline variant on genetic instability that leads to oncogenesis, opening new scenarios for Cohesins' contribution to genetic predisposition to leukemias. [Display omitted] DisclosuresBiondi: Colmmune: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Bluebird: Other: Advisory Board; Novartis: Honoraria.

Nuclear αvβ3 integrin expression, post translational modifications and regulation in hematological malignancies.

αvβ3 integrin, a plasma membrane protein, is amply expressed on an array of tumors. We identified nuclear αvβ3 pool in ovarian cancer cells and were interested to explore this phenomenon in two rare and aggressive types of leukemia, T-cell acute lymphoblastic leukemia (T-ALL) and Mast cell leukemia (MCL) using Jurkat and HMC-1 cell lines, respectively. Moreover, we collected primary cells from patients with chronic lymphocytic leukemia (CLL, n=11), the most common chronic adult leukemia and used human lymphoblastoid cell lines (LCL) generated from normal B cells. Nuclear αvβ3 integrin was assessed by Western blots, confocal microscopy, and the ImageStream technology which combines flow-cytometry with microscopy. We further examined post translational modifications (phosphorylation/glycosylation), nuclear trafficking regulation using inhibitors for MAPK (U0126) and PI3K (LY294002), as well as nuclear interactions by performing Co-immunoprecipitation (Co-IP). αvβ3 integrin was identified in all cell models within the nucleus and is N-glycosylated. In primary CLL cells the β3 integrin monomer is tyrosine Y759 phosphorylated, suggesting an active receptor conformation. MAPK and PI3K inhibition in Jurkat and CLL cells led to αvβ3 enhancement in the nucleus and a reduction in the membrane. The nuclear αvβ3 integrin interacts with ERK, Histone H3 and Lamin B1 in Jurkat, Histone H3 in CLL cells, but not in control LCL cells. To conclude, this observational study provides the identification of nuclear αvβ3 in hematological malignancies and lays the basis for novel cancer-relevant actions, which may be independent from the membrane functions.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR.

CMT is the most common hereditary neuromuscular disorder of the peripheral nervous system with a prevalence of 1/2500 individuals and it is caused by mutations in more than 80 genes. LRSAM1, a RING finger ubiquitin ligase also known as TSG101-associated ligase (TAL), has been associated with Charcot-Marie-Tooth disease type 2P (CMT2P) and to date eight causative mutations have been identified. Little is currently known on the pathogenetic mechanisms that lead to the disease. We investigated the effect of LRSAM1 deregulation on possible LRSAM1 interacting molecules in cell based models. Possible LRSAM1 interacting molecules were identified using protein-protein interaction databases and literature data. Expression analysis of these molecules was performed in both CMT2P patient and control lymphoblastoid cell lines as well as in LRSAM1 and TSG101 downregulated SH-SY5Y cells.TSG101, UBE2N, VPS28, EGFR and MDM2 levels were significantly decreased in the CMT2P patient lymphoblastoid cell line as well as in LRSAM1 downregulated cells. TSG101 downregulation had a significant effect only on the expression of VPS28 and MDM2 and it did not affect the levels of LRSAM1. This study confirms that LRSAM1 is a regulator of TSG101 expression. Furthermore, deregulation of LRSAM1 significantly affects the levels of UBE2N, VPS28, EGFR and MDM2.

Butyrate enhances mitochondrial function during oxidative stress in cell lines from boys with autism

Butyrate (BT) is a ubiquitous short-chain fatty acid (SCFA) principally derived from the enteric microbiome. BT positively modulates mitochondrial function, including enhancing oxidative phosphorylation and beta-oxidation and has been proposed as a neuroprotectant. BT and other SCFAs have also been associated with autism spectrum disorders (ASD), a condition associated with mitochondrial dysfunction. We have developed a lymphoblastoid cell line (LCL) model of ASD, with a subset of LCLs demonstrating mitochondrial dysfunction (AD-A) and another subset of LCLs demonstrating normal mitochondrial function (AD-N). Given the positive modulation of BT on mitochondrial function, we hypothesized that BT would have a preferential positive effect on AD-A LCLs. To this end, we measured mitochondrial function in ASD and age-matched control (CNT) LCLs, all derived from boys, following 24 and 48 h exposure to BT (0, 0.1, 0.5, and 1 mM) both with and without an in vitro increase in reactive oxygen species (ROS). We also examined the expression of key genes involved in cellular and mitochondrial response to stress. In CNT LCLs, respiratory parameters linked to adenosine triphosphate (ATP) production were attenuated by 1 mM BT. In contrast, BT significantly increased respiratory parameters linked to ATP production in AD-A LCLs but not in AD-N LCLs. In the context of ROS exposure, BT increased respiratory parameters linked to ATP production for all groups. BT was found to modulate individual LCL mitochondrial respiration to a common set-point, with this set-point slightly higher for the AD-A LCLs as compared to the other groups. The highest concentration of BT (1 mM) increased the expression of genes involved in mitochondrial fission (PINK1, DRP1, FIS1) and physiological stress (UCP2, mTOR, HIF1α, PGC1α) as well as genes thought to be linked to cognition and behavior (CREB1, CamKinase II). These data show that the enteric microbiome-derived SCFA BT modulates mitochondrial activity, with this modulation dependent on concentration, microenvironment redox state, and the underlying mitochondrial function of the cell. In general, these data suggest that BT can enhance mitochondrial function in the context of physiological stress and/or mitochondrial dysfunction, and may be an important metabolite that can help rescue energy metabolism during disease states. Thus, insight into this metabolic modulator may have wide applications for both health and disease since BT has been implicated in a wide variety of conditions including ASD. However, future clinical studies in humans are needed to help define the practical implications of these physiological findings.

Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events

BackgroundLaboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. This study explored the utility of nanopore sequencing to examine whole BRCA1 mRNA transcripts and to provide accurate categorisation of in-frame and out-of-frame splicing events.MethodsThe exon structure of BRCA1 transcripts from a previously studied control lymphoblastoid cell line were assessed using MinION nanopore sequencing of long-range reverse transcriptase-PCR amplicons.ResultsOur study identified and characterised 32 complete BRCA1 isoforms, including 18 novel isoforms which showed skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as Δ(9,10) and Δ21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Fourteen novel isoforms were formed entirely from a combination of previously identified alternative splice junctions, suggesting that the total number of BRCA1 isoforms might be lower than the number of splicing events reported previously.ConclusionsOur results highlight complexity in BRCA1 transcript structure that has not been described previously. This finding has key implications for predicting the translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.

Oxidative Stress Challenge Uncovers Trichloroacetaldehyde Hydrate-Induced Mitoplasticity in Autistic and Control Lymphoblastoid Cell Lines

Mitoplasticity occurs when mitochondria adapt to tolerate stressors. Previously we hypothesized that a subset of lymphoblastoid cell lines (LCLs) from children with autistic disorder (AD) show mitoplasticity (AD-A), presumably due to previous environmental exposures; another subset of AD LCLs demonstrated normal mitochondrial activity (AD-N). To better understand mitoplasticity in the AD-A LCLs we examined changes in mitochondrial function using the Seahorse XF96 analyzer in AD and Control LCLs after exposure to trichloroacetaldehyde hydrate (TCAH), an in vivo metabolite of the environmental toxicant and common environmental pollutant trichloroethylene. To better understand the role of reactive oxygen species (ROS) in mitoplasticity, TCAH exposure was followed by acute exposure to 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases ROS. TCAH exposure by itself resulted in a decline in mitochondrial respiration in all LCL groups. This effect was mitigated when TCAH was followed by acute DMNQ exposure but this varied across LCL groups. DMNQ did not affect AD-N LCLs, while it neutralized the detrimental effect of TCAH in Control LCLs and resulted in a increase in mitochondrial respiration in AD-A LCLs. These data suggest that acute increases in ROS can activate mitochondrial protective pathways and that AD-A LCLs are better able to activate these protective pathways.

Role of SIRT1/PGC-1α in mitochondrial oxidative stress in autistic spectrum disorder.

Autistic spectrum disorder (ASD) is a neurodevelopmental disorder and has a high prevalence in children. Recently, mitochondrial oxidative stress has been proposed to be associated with ASD. Besides, SIRT1/PGC-1α signaling plays an important role in combating oxidative stress. In this study, we sought to determine the role of SIRT1/PGC-1α signaling in the ASD lymphoblastoid cell lines (LCLs). In this study, the mRNA and protein expressions of SIRT1/PGC-1α axis genes were assessed in 35 children with ASD and 35 healthy controls (matched for age, gender, and IQ). An immortalized LCL was established by transforming lymphocytes with Epstein–Barr virus. Next, we used ASD LCLs and control LCLs to detect SIRT1/PGC-1α axis genes expression and oxidative damage. Finally, the effect of overexpression of PGC-1α on oxidative injury in the ASD LCLs was determined. SIRT1/PGC-1α axis genes expression was downregulated at RNA and protein levels in ASD patients and LCLs. Besides, the translocation of cytochrome c and DIABLO from mitochondria to the cytosol was found in the ASD LCLs. Moreover, the intracellular reactive oxygen species (ROS) and mitochondrial ROS and cell apoptosis were increased in the ASD LCLs. However, overexpression of PGC-1α upregulated the SIRT1/PGC-1α axis genes expression and reduced cytochrome c and DIABLO release in the ASD LCLs. Also, overexpression of PGC-1α reduced the ROS generation and cell apoptosis in the ASD LCLs. Overexpression of PGC-1α could reduce the oxidative injury in the ASD LCLs, and PGC-1α may act as a target for treatment.

Modulation of mitochondrial function by the microbiome metabolite propionic acid in autism and control cell lines.

Propionic acid (PPA) is a ubiquitous short-chain fatty acid, which is a major fermentation product of the enteric microbiome. PPA is a normal intermediate of metabolism and is found in foods, either naturally or as a preservative. PPA and its derivatives have been implicated in both health and disease. Whereas PPA is an energy substrate and has many proposed beneficial effects, it is also associated with human disorders involving mitochondrial dysfunction, including propionic acidemia and autism spectrum disorders (ASDs). We aimed to investigate the dichotomy between the health and disease effects of PPA by measuring mitochondrial function in ASD and age- and gender-matched control lymphoblastoid cell lines (LCLs) following incubation with PPA at several concentrations and durations both with and without an in vitro increase in reactive oxygen species (ROS). Mitochondrial function was optimally increased at particular exposure durations and concentrations of PPA with ASD LCLs, demonstrating a greater enhancement. In contrast, increasing ROS negated the positive PPA effect with the ASD LCLs, showing a greater detriment. These data demonstrate that enteric microbiome metabolites such as PPA can have both beneficial and toxic effects on mitochondrial function, depending on concentration, exposure duration and microenvironment redox state with these effects amplified in LCLs derived from individuals with ASD. As PPA, as well as enteric bacteria, which produce PPA, have been implicated in a wide variety of diseases, including ASD, diabetes, obesity and inflammatory diseases, insight into this metabolic modulator from the host microbiome may have wide applications for both health and disease.

A Restricted Repertoire of De Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.

Haploinsufficiency of RCBTB1 is associated with Coats disease and familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) belongs to a group of genetically and clinically heterogeneous disorders in retinal vascular development. To date, in approximately 50% of patients with FEVR, pathogenic mutations have been detected in FZD4, LRP5, TSPAN12, NDP and ZNF408. In this study, we identified two heterozygous frameshift mutations in RCBTB1 from three Taiwanese cases through exome sequencing. In patient-derived lymphoblastoid cell lines (LCLs), the protein level of RCBTB1 is approximately half that of unaffected control LCLs, which is indicative of a haploinsufficiency mechanism. By employing transient transfection and reporter assays for the transcriptional activity of β-catenin, we demonstrated that RCBTB1 participates in the Norrin/FZD4 signaling pathway and that knockdown of RCBTB1 by shRNA significantly reduced nuclear accumulation of β-catenin under Norrin and Wnt3a treatments. Furthermore, transgenic fli1:EGFP zebrafish with rcbtb1 knockdown exhibited anomalies in intersegmental and intraocular vessels. These results strongly support that reduced RCBTB1 expression may lead to defects in angiogenesis through the Norrin-dependent Wnt pathway, and that RCBTB1 is a putative genetic cause of vitreoretinopathies.

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.

Increased susceptibility to ethylmercury-induced mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines.

The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction.

An Acquired Mutation in Deubiquitinating Enzyme USP11 Associated with Clonal Hematopoiesis in Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (BMFS) affecting one in 105 live births. In the majority of patients the disease results from heterozygous mutations or deletions in genes encoding ribosomal proteins (RP), suggesting haploinsufficiency for RPs causes the disease. DBA presentation and outcome is very variable, even in the same family, with 20% of patients going into remission by an unknown mechanism. In some cases remission in BMFS has been associated with clonal expansion of cells bearing acquired genomic changes. We hypothesized that a similar mechanism may underlie remissions in DBA. In order to detect somatic single nucleotide mutations, copy number variations (CNV), and copy neutral loss of heterozygosity (CN-LOH) in the bone marrow of DBA patients, we took advantage of whole exome sequencing (WES) and single nucleotide polymorphism (SNP) array technologies. Genotyping was performed on paired DNA samples from bone marrow and skin fibroblasts of five independent patients with DBA. About 30 non-synonymous coding differences per sample pair (skin and BM) were called. These were reduced by eliminating 1. mutations due to technical and bioinformatics artifacts that often appear in these data. 2. mutations that were not validated by Sanger sequencing. While analysis and validation is ongoing we have positively identified 2 candidate gene mutations that were found in DNA from BM cells but not from skin fibroblasts from 2 individuals.In the case discussed here a somatic mutation in USP11, a c.G1253A leading to a R418Q amino acid substitution, was detected by WES in a DBA male patient with a relatively mild disease. Sanger sequencing validated this finding in the bone marrow and the peripheral blood of the patient. USP11 is located on the X chromosome and the R418Q substitution is predicted to profoundly affect the catalytic subunit of USP11, according to in-silico analysis.We hypothesized that the USP11 mutation may have favorably affected the patient’s hematopoiesis, leading to a milder form of DBA, due to a compensatory effect on ribosomal biogenesis. To determine the effects of this mutation on RPs we took advantage of lymphoblastoid cell lines (LCLs) from DBA patients. The LCLs of two DBA patients with heterozygous RPL5 mutations were examined along with control LCLs. One of the DBA LCL lines was hemizygous for USP11R418Q and the other was WT at the USP11 locus. The levels of RPL11 and RPL5 were higher in cell lines from the patient hemizygous for USP11R418Q when compared with those from the patient with WT-USP11. Furthermore, levels of RPL5 and RPL11 in the patient carrying the USP11R418Q allele were similar to those of a healthy control. RPL11 is important in regulating the MDM2/p53 pathway implicated in DBA pathogenesis. In a second set of experiments we overexpressed mutant and wild type USP11 in HEK293T cells and observed that ribosomal protein RPL11 was again overexpressed compared with when WT USP11 was mutated. Overexpression of mutant USP11 led to the accumulation of ubiquitinated proteins, suggesting the mutation interfered with deubiquitination.USP11 is known to play a role in DNA repair, the TNF-α and the TGFβ pathways. The precise mechanism of action of USP11 in controlling the level of RPL11 is still obscure. Nevertheless, our findings suggest that USP11 may play an important role in the regulation of RP levels and may be a candidate for investigation as a therapeutic target in DBA. DisclosuresNo relevant conflicts of interest to declare.

De NovoRPS20 Mutations in Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red blood cell aplasia, variable congenital anomalies and increased risk of malignancy. Approximately 50-60% of cases are due to germline, heterozygous mutations in 1 of 13 genes that encode components of either the small (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS29) or large (RPL5, RPL11, RPL15, RPL26, RPL31 and RPL35A) ribosomal subunits. The mutations may be either inherited or de novo. Consistent with DBA being a ribosomopathy, defects in ribosome assembly, altered ribosomal RNA processing and nucleolar stress are observed in cells of patients with DBA and ribosomal protein gene mutations. Mutations in GATA1, a hematopoietic transcription factor, have also been reported in rare X-linked recessive cases.Whole exome sequencing was independently carried out on two white/Hispanic probands with genetically uncharacterized DBA (BMF92 and NCI-62-1). Both presented with transfusion-dependent anemia during the first week of life. Bone marrow evaluations revealed marked erythroid hypoplasia characteristic of DBA. One of the probands (BMF92) experienced a spontaneous remission of his anemia toward the end of the first year of life, possibly related to corticosteroids administered for bronchiolitis. Subsequently, his erythrocyte adenosine deaminase(eADA) level was measured and elevated [10.5 units (mol/min/gm hg); reference range 0.42-3.5 units]. BMF92’s hypoplastic anemia later recurred and was found to be steroid refractory. NCI-62-1 is a male, now 12 years of age and red cell transfusion dependent. BMF92 and NCI-62-1 are of normal stature and have no documented congenital anomalies, although both have experienced chronic colitis. In both cases, family history for DBA was negative and parental eADA levels were within normal limits.Whole exome sequencing of peripheral blood DNA, validated by Sanger or Ion Torrent targeted sequencing, identified heterozygous variants of unknown significance in the ribosomal protein gene RPS20. Parental studies revealed both variants to be de novo. The variants were mutated at the same genomic position, but resulted in different amino acid substitutions in RPS20 [hg19 chr8 56985758, c.251A>T, p.I84N (BMF92) and 56985758, c.251A>C, p.I84S (NCI-62-1)]. Buccal swab analysis performed in BMF92 confirmed the presence of the variant. Both variants were novel, based on inspection of several databases, including dbSNP, 1000 Genomes, NHLBI Exome Sequencing Project, clinical WES data at the Whole Genome Laboratory at Baylor College of Medicine, Kaviar, Human Genome Mutation Database and ClinVar, totally well over 10,000 individuals as well as the COSMIC database, which reports somatic mutations in cancer. In silicoanalyses were consistent with the variants being damaging and disease causing (PolyPhen-2, SIFT and Mutation Taster), affecting highly evolutionarily conserved residues (GERP, PhyloP and Sitewise likelihood-ratio score) and decreasing the stability of the protein structure (MUpro and I-Mutant2.0).The above data strongly suggest that the RPS20 variants in these cases are disease causing. A recent report implicated a germline RPS20 truncating mutation in a four-generation pedigree with familial nonpolyposis colorectal carcinoma; however, the mutation carriers were reported to not manifest features of DBA (Nieminen et al., Gastroenterology 2014). This raises the likelihood of allele-specific effects. Functional assays of our probands’ mutations, including quantification of RPS20 protein and ribosomal RNA precursor steady state levels in patient versus control lymphoblastoid cell lines as well as analysis of the impact of mutant RPS20 protein expression on p53 and downstream targets are underway to determine their contribution to the DBA phenotype. DisclosuresNo relevant conflicts of interest to declare.

Novel insights into the regulatory architecture of CD4+ T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most frequent autoimmune chronic inflammatory disease of the joints and it is characterized by the inflammation of the synovial membrane and the subsequent destruction of the joints. In RA, CD4+ T cells are the main drivers of disease initiation and the perpetuation of the damaging inflammatory process. To date, however, the genetic regulatory mechanisms of CD4+ T cells associated with RA etiology are poorly understood. The genome-wide analysis of expression quantitative trait loci (eQTL) in disease-relevant cell types is a recent genomic integration approach that is providing significant insights into the genetic regulatory mechanisms of many human pathologies. The objective of the present study was to analyze, for the first time, the genome-wide genetic regulatory mechanisms associated with the gene expression of CD4+ T cells in RA. Whole genome gene expression profiling of CD4+ T cells and the genome-wide genotyping (598,258 SNPs) of 29 RA patients with an active disease were performed. In order to avoid the excessive burden of multiple testing associated with genome-wide trans-eQTL analysis, we developed and implemented a novel systems genetics approach. Finally, we compared the genomic regulation pattern of CD4+ T cells in RA with the genomic regulation observed in reference lymphoblastoid cell lines (LCLs). We identified a genome-wide significant cis-eQTL associated with the expression of FAM66C gene (P = 6.51e−9). Using our new systems genetics approach we identified six statistically significant trans-eQTLs associated with the expression of KIAA0101 (P<7.4e−8) and BIRC5 (P = 5.35e−8) genes. Finally, comparing the genomic regulation profiles between RA CD4+ T cells and control LCLs we found 20 genes showing differential regulatory patterns between both cell types. The present genome-wide eQTL analysis has identified new genetic regulatory elements that are key to the activity of CD4+ T cells in RA.