NIPBL Mutations Research Articles

Synthetic Lethality between Cohesin and WNT Signaling Pathways in Diverse Cancer Contexts.

Cohesin is a highly conserved ring-shaped complex involved in topologically embracing chromatids, gene expression regulation, genome compartmentalization, and genome stability maintenance. Genomic analyses have detected mutations in the cohesin complex in a wide array of human tumors. These findings have led to increased interest in cohesin as a potential target in cancer therapy. Synthetic lethality has been suggested as an approach to exploit genetic differences in cancer cells to influence their selective killing. In this study, we show that mutations in ESCO1, NIPBL, PDS5B, RAD21, SMC1A, SMC3, STAG2, and WAPL genes are synthetically lethal with stimulation of WNT signaling obtained following LY2090314 treatment, a GSK3 inhibitor, in several cancer cell lines. Moreover, treatment led to the stabilization of β-catenin and affected the expression of c-MYC, probably due to the occupancy decrease in cohesin at the c-MYC promoter. Finally, LY2090314 caused gene expression dysregulation mainly involving pathways related to transcription regulation, cell proliferation, and chromatin remodeling. For the first time, our work provides the underlying molecular basis for synthetic lethality due to cohesin mutations and suggests that targeting the WNT may be a promising therapeutic approach for tumors carrying mutated cohesin.

Nipbl Haploinsufficiency Leads to Delayed Outflow Tract Septation and Aortic Valve Thickening.

Cornelia de Lange Syndrome (CdLS) patients, who frequently carry a mutation in NIPBL, present an increased incidence of outflow tract (OFT)-related congenital heart defects (CHDs). Nipbl+/- mice recapitulate a number of phenotypic traits of CdLS patients, including a small body size and cardiac defects, but no study has specifically focused on the valves. Here, we show that adult Nipbl+/- mice present aortic valve thickening, a condition that has been associated with stenosis. During development, we observed that OFT septation and neural crest cell condensation was delayed in Nipbl+/- embryos. However, we did not observe defects in the deployment of the main lineages contributing to the semilunar valves. Indeed, endocardial endothelial-to-mesenchymal transition (EndMT), analysed via outflow tract explants, and neural crest migration, analysed via genetic lineage tracing, did not significantly differ in Nipbl+/- mice and their wild-type littermates. Our study provides the first direct evidence for valve formation defects in Nipbl+/- mice and points to specific developmental defects as an origin for valve disease in patients.

Establishment of a human induced pluripotent stem cell line, KMUGMCi002-A, from a patient bearing a heterozygous c.6362_6364del mutation in the NIPBL gene leading Cornelia de Lange syndrome (CdLS)

Cornelia de Lange syndrome (CdLS) is a multiple congenital anomalies syndrome caused by mutations in the cohesion complex. The mutations in NIPBL, one of cohesion regulatory proteins, are the most frequent cause of CdLS. The peripheral blood mononuclear cells (PBMCs) from a patient carrying a heterozygous 3 bp deletion in Exon 37 of the NIPBL gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The deleted mutation in NIPBL will cause the abnormal truncated protein, which is known to associated with CdLS. The established human induced pluripotent cell (hiPSC) line will enable proper in vitro disease modelling of CdLS.Resource Table:

Cohesin Mutations Induce Chromatin Conformation Perturbation of the H19/IGF2 Imprinted Region and Gene Expression Dysregulation in Cornelia de Lange Syndrome Cell Lines.

Traditionally, Cornelia de Lange Syndrome (CdLS) is considered a cohesinopathy caused by constitutive mutations in cohesin complex genes. Cohesin is a major regulator of chromatin architecture, including the formation of chromatin loops at the imprinted IGF2/H19 domain. We used 3C analysis on lymphoblastoid cells from CdLS patients carrying mutations in NIPBL and SMC1A genes to explore 3D chromatin structure of the IGF2/H19 locus and evaluate the influence of cohesin alterations in chromatin architecture. We also assessed quantitative expression of imprinted loci and WNT pathway genes, together with DMR methylation status of the imprinted genes. A general impairment of chromatin architecture and the emergence of new interactions were found. Moreover, imprinting alterations also involved the expression and methylation levels of imprinted genes, suggesting an association among cohesin genetic defects, chromatin architecture impairment, and imprinting network alteration. The WNT pathway resulted dysregulated: canonical WNT, cell cycle, and WNT signal negative regulation were the most significantly affected subpathways. Among the deregulated pathway nodes, the key node of the frizzled receptors was repressed. Our study provides new evidence that mutations in genes of the cohesin complex have effects on the chromatin architecture and epigenetic stability of genes commonly regulated by high order chromatin structure.

Genomic characterization of relapsed acute myeloid leukemia reveals novel putative therapeutic targets

AbstractRelapse is the leading cause of death of adult and pediatric patients with acute myeloid leukemia (AML). Numerous studies have helped to elucidate the complex mutational landscape at diagnosis of AML, leading to improved risk stratification and new therapeutic options. However, multi–whole-genome studies of adult and pediatric AML at relapse are necessary for further advances. To this end, we performed whole-genome and whole-exome sequencing analyses of longitudinal diagnosis, relapse, and/or primary resistant specimens from 48 adult and 25 pediatric patients with AML. We identified mutations recurrently gained at relapse in ARID1A and CSF1R, both of which represent potentially actionable therapeutic alternatives. Further, we report specific differences in the mutational spectrum between adult vs pediatric relapsed AML, with MGA and H3F3A p.Lys28Met mutations recurrently found at relapse in adults, whereas internal tandem duplications in UBTF were identified solely in children. Finally, our study revealed recurrent mutations in IKZF1, KANSL1, and NIPBL at relapse. All of the mentioned genes have either never been reported at diagnosis in de novo AML or have been reported at low frequency, suggesting important roles for these alterations predominantly in disease progression and/or resistance to therapy. Our findings shed further light on the complexity of relapsed AML and identified previously unappreciated alterations that may lead to improved outcomes through personalized medicine.

The role of NIPBL during cardiac progenitor specification

Congenital heart disease can result from defects in the epigenetic events driving cardiogenesis. Little is currently known about the chromatin structural changes that occur in cardiac progenitors. We are focusing on the role of NIPBL, a cohesin complex loading partner that plays a key role in the regulation of chromatin structure, during cardiogenesis. NIPBL mutations are associated with Cornelia de Lange Syndrome, characterised by an array of cardiac defects. To characterise the role of Nipbl during cardiogenesis. We have used scRNAseq to analyse transcriptional alterations resulting from conditional knockout of NIPBL in early mouse embryos. Complementary ChIP-seq experiments are allowing us to characterise the epigenetic features of genes whose expression is altered as a result of NIPBL deficiency. scRNAseq has revealed that NIPBL deficiency leads to dramatic alterations in several transcripts, including members of the TGFbeta signalling pathway, within well-defined cardiac progenitor populations. These genes are found in regions of highly dynamic chromatin. Furthermore, specification of progenitor populations, including the lateral plate mesoderm, is affected. The identification of genes whose expression is highly sensitive to NIPBL levels in cardiac progenitors has revealed potential therapeutic targets for congenital heart defects in Cornelia de Lange Syndrome patients. Also, this work provides novel insights into the role of NIPBL/cohesin during cardiogenesis.

A report of 2 cases of Cornelia de Lange syndrome (CdLS) and an analysis of clinical and genetic characteristics in a Chinese CdLS cohort.

Background Cornelia de Lange syndrome (CdLS) is a rare dominantly inherited developmental disorder with an estimated prevalence of 0.5-10:100,000 and no racial disparity in prevalence. The aim of this study was to present two unrelated Chinese CdLS individuals with mutations in NIPBL and to perform a comprehensive analysis of a Chinese cohort with CdLS. Subjects and methods Two unrelated Chinese patients complaining of short stature were referred to the outpatient department of Peking Union Medical College Hospital (PUMCH). Their clinical data at birth and at the most recent assessment were collected. Mutation analysis was carried out by whole exome sequencing. Twenty-four Chinese cases with CdLS were identified through a systematic review of the literature published between 1987 and 2017. Results Two patients presented with typical phenotypes, characteristic complications of CdLS and mutations in the NIPBL gene. The average age at diagnosis of the 26 Chinese cases was higher than that of other cohorts. The frequencies of characteristic manifestations of CdLS were similar with those of other populations. Conclusions By investigating 26 Chinese cases of CdLS, we observed that the clinical data and gene variants in the Chinese cohort of CdLS patients were generally in accordance with those of other populations.

Identification and prenatal diagnosis of a novel NIPBL mutation underlying Cornelia De Lange syndrome

To explore the genetic basis for an infant featuring developmental delay, hand deformity and hypertonia of extremities. Clinical data and peripheral blood samples of the proband and her parents were collected. Following DNA extraction, potential mutations were screened on an Ion PGM platform using a gene panel. Suspected mutation was verified by PCR and Sanger sequencing. A novel heterozygous nonsense mutation, c.2521C>T(p.R841X), was identified in the NIPBL gene. The mutation may cause premature termination of translation of the adhesion protein loading factor at 841st amino acids. The same mutation was not found in her parents and 931 healthy controls, and was absent from public databases including ExAC and 1000G. Bioinformatic analysis suggested the mutation to be disease causing. The c.2521C>T (p.R841X) mutation of the NIPBL gene probably underlies the Cornelia De Lange syndrome in the infant. Prenatal diagnosis may be provided to this family upon their subsequent pregnancy.

The Cornelia de Lange Syndrome-associated factor NIPBL interacts with BRD4 ET domain for transcription control of a common set of genes

Mutations in NIPBL are the major cause of Cornelia de Lange Syndrome (CdLS). NIPBL is the cohesin-loading factor and has recently been associated with the BET (bromodomains and extra-terminal (ET) domain) proteins BRD2 and BRD4. Related to this, a CdLS-like phenotype has been described associated to BRD4 mutations. Here, we show direct interaction of NIPBL with different BET members in yeast, and selective interaction with BRD4 in cells, being the ET domain involved in the interaction. To understand the relationship between NIPBL and BET proteins, we have performed RNA-Seq expression analysis following depletion of the different proteins. Results indicate that genes regulated by NIPBL largely overlap with those regulated by BRD4 but not with those regulated by BRD2. ChIP-Seq analysis indicates preferential NIPBL occupancy at promoters, and knockdown experiments show mutual stabilization of NIPBL and BRD4 on co-regulated promoters. Moreover, human fibroblasts from CdLS probands with mutations in NIPBL show reduced BRD4 at co-occupied promoters. Functional analysis in vivo, using mutants of Drosophila melanogaster, confirmed the genetic interaction between Nipped-B and fs(1)h, the orthologs of human NIPBL and BRD4, respectively. Thus, we provide evidence for NIPBL and BRD4 cooperation in transcriptional regulation, which should contribute to explain the recently observed CdLS-like phenotype associated with BRD4 mutations.

Next generation sequencing identified two novel mutations in NIPBL and a frame shift mutation in CREBBP in three Chinese children

BackgroundCornelia de Lange syndrome (CdLS) and Rubinstein-Taybi syndrome (RSTS) are both rare congenital multiple malformation disorders caused by genes associated with transcription. They share a number of similar features clinically. In addition, it is difficult to make a molecular diagnosis rapidly and detect the mosaic mutation when only sanger sequencing is taken. This study aims to report three novel mutations in three Chinese children identified by next generation sequencing.ResultsWe describe patient 1 and patient 2 presenting with characteristics of CdLS with mutations in NIPBL and patient 3 with a frame shift mutation in CREBBP who can be diagnosed as RSTS clinically and also have similar symptoms with CdLS to some extent. The splicing site c.4321-1G > A transversion in NIPBL is a mosaic mutation and produces an abnormal transcript bearing the loss of exon 20. The nonsense mutation c.218C > A in NIPBL and the frame shift c.1715delC mutation in CREBBP generate stop codon and yield the premature termination of proteins.ConclusionsIn general, we detect three novel heterozygous mutations including a splicing mutation and a nonsense mutation in NIPBL and a frame shift in CREBBP. And several similar features observed in patients indicate the clinical complexity and clinically overlapping of CdLS and RSTS termed “transcriptomopathies”, suggest the underlying molecular mechanism and emphasize the utilization of next generation sequencing technologies.

NIPBL+/\u2212 haploinsufficiency reveals a constellation of transcriptome disruptions in the pluripotent and cardiac states

Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in NIPBL are responsible for the majority of cases of CdLS. Through RNA-sequencing of human induced pluripotent stem cells and in vitro-derived cardiomyocytes, we identified hundreds of mRNAs, pseudogenes, and non-coding RNAs with altered expression in NIPBL+/− patient-derived cells. We demonstrate that NIPBL haploinsufficiency leads to upregulation of gene sets identified in functions related to nucleosome, chromatin assembly, RNA modification and downregulation of Wnt signaling, cholesterol biosynthesis and vesicular transport in iPSC and cardiomyocytes. Mutations in NIPBL result in the dysregulation of many genes responsible for normal heart development likely resulting in the variety of structural cardiac defects observed in the CdLS population.

Genotype-phenotype correlations in Cornelia de Lange syndrome: Behavioral characteristics and changes with age.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder associated with unusual facial features, limb abnormalities, a wide range of health conditions, and intellectual disability. Mutations in five genes that encode (SMC1A, SMC3, RAD21) or regulate (NIPBL, HDAC8) the cohesin complex have been identified in up to 70% of individuals. Genetic cause remains unknown for a proportion of individuals. There is substantial heterogeneity in all aspects of CdLS but very little is known about what predicts phenotypic heterogeneity. In this study, we evaluated genotype-phenotype associations in 34 individuals with CdLS. Participants with NIPBL mutations had significantly lower self help skills and were less likely to have verbal skills relative to those who were negative for the NIPBL mutation. No significant differences were identified between the groups in relation to repetitive behavior, mood, interest and pleasure, challenging behavior, activity, impulsivity, and characteristics of autism spectrum disorder whilst controlling differences in self help skills. Significant correlations indicating lower mood, interest and pleasure, and increased insistence on sameness with older age were identified for those who were NIPBL mutation positive. The findings suggest similarities in the behavioral phenotype between those with and without the NIPBL mutation once differences in self help skills are controlled for. However, there may be subtle differences in the developmental trajectory of these behaviors according to genetic mutation status in CdLS.

Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

SummaryMutations in NIPBL are the most frequent cause of Cornelia de Lange syndrome (CdLS), a developmental disorder encompassing several neurological defects, including intellectual disability and seizures. How NIPBL mutations affect brain development is not understood. Here we identify Nipbl as a functional interaction partner of the neural transcription factor Zfp609 in brain development. Depletion of Zfp609 or Nipbl from cortical neural progenitors in vivo is detrimental to neuronal migration. Zfp609 and Nipbl overlap at genomic binding sites independently of cohesin and regulate genes that control cortical neuron migration. We find that Zfp609 and Nipbl interact with the Integrator complex, which functions in RNA polymerase 2 pause release. Indeed, Zfp609 and Nipbl co-localize at gene promoters containing paused RNA polymerase 2, and Integrator similarly regulates neuronal migration. Our data provide a rationale and mechanistic insights for the role of Nipbl in the neurological defects associated with CdLS.

The Hole and the Whole: Lessons from Manipulation of Nipbl Deficiency.

Congenital heart defects (CHDs) affect 2%–3% of newborns and remain challenging clinically. There is an ongoing project to elucidate the causes of CHDs, focusing primarily on genetics as dictated by the epidemiology. In a paper published in this issue, Santos and colleagues describe studies of Cornelia de Lange syndrome-associated secundum atrial septal defects (ASDs) caused by NIPBL mutations, undertaken with a targeted trapping allele in mice. They show that Nipbl haploinsufficiency in either of two cell populations was sufficient to engender ASDs but that expression solely in either one of those populations was sufficient to rescue them. This work provides novel insights into incomplete penetrance and oligogenic effects underlying CHDs.

Special cases in Cornelia de Lange syndrome: The Spanish experience.

Cornelia de Lange Syndrome (CdLS) is an autosomal dominant (NIPBL, SMC3, and RAD21) or X-linked (SMC1A and HDAC8) disorder, characterized by distinctive craniofacial appearance, growth retardation, intellectual disability, and limb anomalies. In 2005, the Spanish CdLS Reference Center was started and now we have more than 270 cases in our database. In this special issue, we describe some of the unique or atypical patients studied by our group, whose clinical features have contributed to the expansion of the CdLS classical phenotype, helping clinicians to diagnose it. We include the case of a male with unilateral tibial hypoplasia and peroneal agenesis who had a mutation in NIPBL; we also describe one patient with a mutation in NIPBL and somatic mosaicism identified by new generation sequencing techniques; we also include one patient with CdLS and Turner syndrome; and last, an interesting patient with a duplication of the SMC1A gene. Finally, we make a short review of the splicing mutations we have found in NIPBL regarding the new knowledge on the physiological variants of the gene. © 2016 Wiley Periodicals, Inc.

NIPBL expression levels in CdLS probands as a predictor of mutation type and phenotypic severity.

Cornelia de Lange syndrome (CdLS) is a rare, genetically heterogeneous multisystem developmental disorder with a high degree of variability in its clinical presentation. Approximately 65% of probands harbor mutations in genes that encode core components (SMC1A, SMC3, and RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex, of which mutations in NIPBL are the most common. Cohesin plays a canonical role in sister chromatid cohesion during cell division and non-canonical roles in DNA repair, stem cell maintenance and differentiation, and regulation of gene expression. Disruption of the latter role seems to be the major contributor to the underlying molecular pathogenesis of CdLS. NIPBL is required for loading and unloading the cohesin complex onto chromosomes. The expression levels of NIPBL itself appear to be tightly regulated and highly evolutionarily conserved. Droplet digital PCR was used to quantify NIPBL mRNA expression levels with high precision from a cohort of 37 samples (NIPBL, SMC1A, SMC3, and HDAC8 mutation positive probands and negative control). Probands with severe forms of CdLS or severe mutation types were found to have lower levels of NIPBL in comparison to phenotypically milder patients and controls. Levels of NIPBL also correlated with the presence of mutations in different CdLS-causing genes. The data suggests that NIPBL levels are closely correlated with the severity of CdLS and with specific causative genes and types of mutations. ddPCR may provide a tool to assist in diagnostic approaches to CdLS, for genetic counseling and prognosis, and for monitoring potential therapeutic modalities in the future. © 2016 Wiley Periodicals, Inc.

Characterization of limb differences in children with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a well-described multisystem developmental disorder characterized by dysmorphic facial features, growth and behavioral deficits, and cardiac, gastrointestinal, and limb anomalies. The limb defects seen in CdLS can be mild, with small feet or hands only, or can be severe, with variable deficiency defects involving primarily the ulnar structures and ranging from mild hypoplasia of the fifth digit to complete absence of the forearm. Interestingly, the upper limbs are typically much more involved than the lower extremities that generally manifest with small feet and 2-3 syndactyly of the toes and shortened fourth metatarsal. The upper limbs often manifest asymmetric involvement. The limb findings in our cohort of 378 individuals with CdLS demonstrate a consistent pattern of laterality and symmetry involvement (with increased severity of right-sided limb in individuals with asymmetric limb defects) and a correlation of more significant limb defects with an increased risk of other structural anomalies, and more severe behavioral outcomes. Additionally, we found that individuals with mutations in NIPBL were most likely to have limb defects compared to mutations in other genes with nonsense, exonic deletion, and frameshift mutations being most prevalent in those with limb defects. Characterization of the limb differences in children with CdLS may provide a tool to assist in genetic counseling and determining prognosis. This paper will review the limb involvement in a large cohort of individuals with CdLS assessing the correlation with molecular etiologies, symmetry, additional structural birth defects, and cognitive outcomes. © 2016 Wiley Periodicals, Inc.

A series of 38 novel germline and somatic mutations of NIPBL in Cornelia de Lange syndrome.

Cornelia de Lange syndrome is a multisystemic developmental disorder mainly related to de novo heterozygous NIPBL mutation. Recently, NIPBL somatic mosaicism has been highlighted through buccal cell DNA study in some patients with a negative molecular analysis on leukocyte DNA. Here, we present a series of 38 patients with a Cornelia de Lange syndrome related to a heterozygous NIPBL mutation identified by Sanger sequencing. The diagnosis was based on the following criteria: (i) intrauterine growth retardation and postnatal short stature, (ii) feeding difficulties and/or gastro-oesophageal reflux, (iii) microcephaly, (iv) intellectual disability, and (v) characteristic facial features. We identified 37 novel NIPBL mutations including 34 in leukocytes and 3 in buccal cells only. All mutations shown to have arisen de novo when parent blood samples were available. The present series confirms the difficulty in predicting the phenotype according to the NIPBL mutation. Until now, somatic mosaicism has been observed for 20 cases which do not seem to be consistently associated with a milder phenotype. Besides, several reports support a postzygotic event for those cases. Considering these elements, we recommend a first-line buccal cell DNA analysis in order to improve gene testing sensitivity in Cornelia de Lange syndrome and genetic counselling.

Recognition of the Cornelia de Lange syndrome phenotype with facial dysmorphology novel analysis.

Facial analysis systems are becoming available to healthcare providers to aid in the recognition of dysmorphic phenotypes associated with a multitude of genetic syndromes. These technologies automatically detect facial points and extract various measurements from images to recognize dysmorphic features and evaluate similarities to known facial patterns (gestalts). To evaluate such systems' usefulness for supporting the clinical practice of healthcare professionals, the recognition accuracy of the Cornelia de Lange syndrome (CdLS) phenotype was examined with FDNA's automated facial dysmorphology novel analysis (FDNA) technology. In the first experiment, 2D facial images of CdLS patients with either an NIPBL or SMC1A gene mutation as well as non-CdLS patients which were assessed by dysmorphologists in a previous study were evaluated by the FDNA technology; the average detection rate of experts was 77% while the system's detection rate was 87%. In the second study, when a new set of NIPBL, SMC1A and non-CdLS patient photos was evaluated, the detection rate increased to 94%. The results from both studies indicated that the system's detection rate was comparable to that of dysmorphology experts. Therefore, utilizing such technologies may be a useful tool in a clinical setting.

NIPBL Controls RNA Biogenesis to Prevent Activation of the Stress Kinase PKR.

SUMMARYNIPBL, a cohesin loader, has been implicated in transcriptional control and genome organization. Mutations in NIPBL, cohesin, and its deacetylase HDAC8 result in Cornelia de Lange syndrome. We report activation of the RNA-sensing kinase PKR in human lymphoblastoid cell lines carrying NIPBL or HDAC8 mutations, but not SMC1A or SMC3 mutations. PKR activation can be triggered by unmodified RNAs. Gene expression profiles in NIPBL-deficient lymphoblastoid cells and mouse embryonic stem cells reveal lower expression of genes involved in RNA processing and modification. NIPBL mutant lymphoblastoid cells show reduced proliferation and protein synthesis with increased apoptosis, all of which are partially reversed by a PKR inhibitor. Non-coding RNAs from an NIPBL mutant line had less m6A modification and activated PKR activity in vitro. This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation.