Identification and Functional Characterization of Two Intronic<i>NIPBL</i>Mutations in Two Patients with Cornelia de Lange Syndrome

Teresa-Rodrigo, María Esperanza; Eckhold, Juliane; Puisac, Beatriz; Pozojevic, Jelena; Parenti, Ilaria; Baquero-Montoya, Carolina; Gil-Rodríguez, María Concepción; Braunholz, Diana; Dalski, Andreas; Hernández-Marcos, María; Ariadna, Ayerza; Bernal, María Luisa; Ramos, Feliciano J.; Wieczorek, Dagmar; Gillessen‐Kaesbach, Gabriele; Pié, Juan; Kaiser, Frank J.

doi:10.1155/2016/8742939

Cited by 13 publications

(15 citation statements)

References 31 publications

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“…To date, genetic alterations in NIPBL have been detected in approximately 70% of CdLS patients (OMIM: 608667) ( Nizon et al, 2016 ). Genetic variants affecting gene splicing account for 15% of NIPBL mutations ( Mannini et al, 2013 ; Teresa-Rodrigo et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

Mosaic Intronic NIPBL Variant in a Family With Cornelia de Lange Syndrome

et al. 2018

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Cornelia de Lange Syndrome (CdLS) is a well described multiple malformation syndrome caused by alterations in genes encoding subunits or regulators of the cohesin complex. In approximately 70% of CdLS patients, pathogenic NIPBL variants are detected and 15% of them are predicted to affect splicing. Moreover, a large portion of genetic variants in NIPBL was shown to be somatic mosaicism. Here we report two family members with different expression of the CdLS phenotype. In both individuals, a c.869-2A>G (r.869_1495del) substitution was detected, affecting a conserved splice-acceptor site. Deep sequencing revealed the presence of somatic mosaicism in the mother. The substitution was detected in 23% of the sequencing reads using DNA derived from blood samples and 51% in DNA from buccal swabs. The analysis of blood DNA of the son excluded the presence of somatic mosaicism. Correlation of molecular and clinical data revealed that various distribution of genetic alteration in different cell types had an impact on the expression of observed clinical features in both individuals.

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Section: Introductionmentioning

confidence: 99%

Mosaic Intronic NIPBL Variant in a Family With Cornelia de Lange Syndrome

et al. 2018

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“…Consistent with previous reports [ 20 , 21 ], the NIPBL mRNA levels in the patient cells were reduced to 60–70% ( Fig 5B and S8B Fig ). Explanations for this imply either a downregulation of the NIPBL gene and/or a degradation of the NIPBL transcript by the nonsense-mediated mRNA degradation pathway, as has been previously hypothesized [ 61 ]. In the first case we might expect a misregulation of NIPBL-AS1 since our data show that the transcription of NIPBL and NIPBL-AS1 are interconnected.…”

Section: Resultsmentioning

confidence: 86%

Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element

et al. 2017

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Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA.Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS. Author summaryThe most frequent mutations in the human developmental disorder Cornelia de Lange Syndrome (CdLS) occur in the NIPBL gene. NIPBL is critical for chromatin-association of the cohesin complex and has a dual role as transcription factor. The regulation of the NIPBL gene is of great interest since organisms are very sensitive to NIPBL levels. For instance, severely affected patients showed only~65% and mildly affected patients~75% of NIPBL mRNA levels. One case reports a CdLS phenotype with as little as 15% reduction of the NIPBL transcript. Further, in a number of patients with CdLS phenotype no mutations in known CdLS genes are found, raising the question whether mutations could also occur in gene-regulatory elements. Here we investigate the role of a long non-coding RNA NIPBL-AS1 originating from a promoter shared with NIPBL and observe a co-regulation by a distal enhancer that we have identified and that seems to be conserved between tissues. Deletion of the enhancer by CRISPR leads to reduced expression of NIPBL and NIPBL-AS1 but also of NIPBL target genes that were found to be dysregulated in CdLS patient cells. The lncRNA NIPBL-AS1 itself has no role for NIPBL expression, but its transcription reduces the expression of the NIPBL gene and vice versa, thus revealing an interesting mechanism for the fine-tuning of expression levels.

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“…NIPBL expression was assessed with two different antibodies, one raised against its N-terminus and one recognizing its C-terminus. Notably, mutant NIPBL transcripts partially Page 6 on 47 undergo nonsense-mediated mRNA decay in cell lines of patients with heterozygous truncating variants in NIPBL; consequently, the corresponding truncated protein is unlikely to be detected 32,33 .…”

Section: Cells With a Homozygous Loss-of-function Mutation In Nipbl Ementioning

confidence: 99%

MAU2 and NIPBL variants in Cornelia de Lange syndrome reveal MAU2-independent loading of cohesin and uncover a protective mechanism against early truncating mutations in NIPBL

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et al. 2018

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Cornelia de Lange syndrome (CdLS) is a rare developmental disorder caused by mutations in genes related to the cohesin complex. For its association with chromatin, cohesin depends on a heterodimer formed by NIPBL and MAU2, which interact via their respective N-termini. Variants in NIPBL are the main cause of CdLS and result in NIPBL haploinsufficiency.Using CRISPR, we generated cells homozygous for an out-of-frame duplication in NIPBL.Remarkably, alternative translation initiation rescued NIPBL expression in these cells and produced an N-terminally truncated NIPBL that lacks MAU2-interaction domain, causing a dramatic reduction of MAU2 protein levels. Strikingly, this protective mechanism allows nearly normal amounts of cohesin to be loaded onto chromatin in a manner that is independent of functional NIPBL/MAU2 complexes and therefore in contrast to previous findings.We also report the first pathogenic variant in MAU2, a deletion of seven amino acids important for wrapping the N-terminus of NIPBL within MAU2. The mutation causes dramatic reduction of MAU2 heterodimerization with NIPBL, hence undermining the stability of both proteins.Our data confirm NIPBL haploinsufficiency as the major pathogenic mechanism of CdLS and give new insights into the molecular mechanisms responsible for this neurodevelopmental disorder. Our work also unveils an alternative translation-based mechanism that protects cells from out-of-frame variants of NIPBL and that may be of relevance in other genetic conditions.

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Identification and Functional Characterization of Two IntronicNIPBLMutations in Two Patients with Cornelia de Lange Syndrome

Cited by 13 publications

References 31 publications

Mosaic Intronic NIPBL Variant in a Family With Cornelia de Lange Syndrome

Mosaic Intronic NIPBL Variant in a Family With Cornelia de Lange Syndrome

Regulation of the cohesin-loading factor NIPBL: Role of the lncRNA NIPBL-AS1 and identification of a distal enhancer element

MAU2 and NIPBL variants in Cornelia de Lange syndrome reveal MAU2-independent loading of cohesin and uncover a protective mechanism against early truncating mutations in NIPBL

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