Applicability of liquid biopsies to represent the mutational profile of tumor tissue from different cancer entities.

Genetic investigation of tumor heterogeneity and clonal evolution in solid cancers could be assisted by the analysis of liquid biopsies. However, tumors of various entities might release different quantities of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) into the bloodstream, potentially limiting the diagnostic potential of liquid biopsy in distinct tumor histologies. Patients with advanced colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma (MEL) were enrolled in the study, representing tumors with different metastatic patterns. Mutation profiles of cfDNA, CTCs, and tumor tissue were assessed by panel sequencing, targeting 327 cancer-related genes. In total, 30 tissue, 18 cfDNA, and 7 CTC samples from 18 patients were sequenced. Best concordance between the mutation profile of tissue and cfDNA was achieved in CRC and MEL, possibly due to the remarkable heterogeneity of HNSCC (63%, 55% and 11%, respectively). Concordance especially depended on the amount of cfDNA used for library preparation. While 21 of 27 (78%) tissue mutations were retrieved in high-input cfDNA samples (30-100 ng, N = 8), only 4 of 65 (6%) could be detected in low-input samples (<30 ng, N = 10). CTCs were detected in 13 of 18 patients (72%). However, downstream analysis was limited by poor DNA quality, allowing targeted sequencing of only seven CTC samples isolated from four patients. Only one CTC sample reflected the mutation profile of the respective tumor. Private mutations, which were detected in CTCs but not in tissue, suggested the presence of rare subclones. Our pilot study demonstrated superiority of cfDNA- compared to CTC-based mutation profiling. It was further shown that CTCs may serve as additional means to detect rare subclones possibly involved in treatment resistance. Both findings require validation in a larger patient cohort.

Preclinical models of head and neck squamous cell carcinoma for a basic understanding of cancer biology and its translation into efficient therapies.

Comprehensive molecular characterization of head and neck squamous cell carcinoma (HNSCC) has led to the identification of distinct molecular subgroups with fundamental differences in biological properties and clinical behavior. Despite improvements in tumor classification and increased understanding about the signaling pathways involved in neoplastic transformation and disease progression, current standard-of-care treatment for HNSCC mostly remains to be based on a stage-dependent strategy whereby all patients at the same stage receive the same treatment. Preclinical models that closely resemble molecular HNSCC subgroups that can be exploited for dissecting the biological function of genetic variants and/or altered gene expression will be highly valuable for translating molecular findings into improved clinical care. In the present review, we merge and discuss existing and new information on established cell lines, primary two- and three-dimensional ex vivo tumor cultures from HNSCC patients, and animal models. We review their value in elucidating the basic biology of HNSCC, molecular mechanisms of treatment resistance and their potential for the development of novel molecularly stratified treatment.

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

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.

Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.

Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome.

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

Exome sequencing unravels unexpected differential diagnoses in individuals with the tentative diagnosis of Coffin-Siris and Nicolaides-Baraitser syndromes.

Coffin-Siris syndrome (CSS) and Nicolaides-Baraitser syndrome (NCBRS) are rare intellectual disability/congenital malformation syndromes that represent distinct entities but show considerable clinical overlap. They are caused by mutations in genes encoding members of the BRG1- and BRM-associated factor (BAF) complex. However, there are a number of patients with the clinical diagnosis of CSS or NCBRS in whom the causative mutation has not been identified. In this study, we performed trio-based whole-exome sequencing (WES) in ten previously described but unsolved individuals with the tentative diagnosis of CSS or NCBRS and found causative mutations in nine out of ten individuals. Interestingly, our WES analysis disclosed overlapping differential diagnoses including Wiedemann-Steiner, Kabuki, and Adams-Oliver syndromes. In addition, most likely causative de novo mutations were identified in GRIN2A and SHANK3. Moreover, trio-based WES detected SMARCA2 and SMARCA4 deletions, which had not been annotated in a previous Haloplex target enrichment and next-generation sequencing of known CSS/NCBRS genes emphasizing the advantages of WES as a diagnostic tool. In summary, we discuss the phenotypic and diagnostic challenges in clinical genetics, establish important differential diagnoses, and emphasize the cardinal features and the broad clinical spectrum of BAF complex disorders and other disorders caused by mutations in epigenetic landscapers.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼ 1%-2% of CdLS-like phenotypes.

Hidden mutations in Cornelia de Lange syndrome limitations of sanger sequencing in molecular diagnostics.

Cornelia de Lange syndrome (CdLS) is a well-characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.

THAP1, the gene mutated in DYT6 dystonia, autoregulates its own expression.

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably, mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly, expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here, we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction, luciferase reporter gene assays, and (quantitative) chromatin immunoprecipitation (ChIP), we defined the THAP1 minimal promoter to a 480bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe, Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably, endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast, higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus, we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers.

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

A novel missense mutation in CACNA1A evaluated by in silico protein modeling is associated with non-episodic spinocerebellar ataxia with slow progression.

Spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are allelic disorders of the gene CACNA1A encoding the P/Q subunit of a voltage gated calcium channel. While SCA6 is related to repeat expansions affecting the C-terminal part of the protein, EA2 and FHM phenotypes are usually associated with nonsense and missense mutations leading to impaired channel properties. In three unrelated families with dominant cerebellar ataxia, symptoms cosegregated with CACNA1A missense mutations of evolutionary highly conserved amino acids (exchanges p.E668K, p.R583Q and p.D302N). To evaluate pathogenic effects, in silico, protein modeling analyses were performed which indicate structural alterations of the novel mutation p.E668K within the homologous domain 2 affecting CACNA1A protein function. The phenotype is characterised by a very slowly progressive ataxia, while ataxic episodes or migraine are uncommon. These findings enlarge the phenotypic spectrum of CACNA1A mutations.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as ß1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

RAD21 mutations cause a human cohesinopathy.

The evolutionarily conserved cohesin complex was originally described for its role in regulating sister-chromatid cohesion during mitosis and meiosis. Cohesin and its regulatory proteins have been implicated in several human developmental disorders, including Cornelia de Lange (CdLS) and Roberts syndromes. Here we show that human mutations in the integral cohesin structural protein RAD21 result in a congenital phenotype consistent with a "cohesinopathy." Children with RAD21 mutations display growth retardation, minor skeletal anomalies, and facial features that overlap findings in individuals with CdLS. Notably, unlike children with mutations in NIPBL, SMC1A, or SMC3, these individuals have much milder cognitive impairment than those with classical CdLS. Mechanistically, these mutations act at the RAD21 interface with the other cohesin proteins STAG2 and SMC1A, impair cellular DNA damage response, and disrupt transcription in a zebrafish model. Our data suggest that, compared to loss-of-function mutations, dominant missense mutations result in more severe functional defects and cause worse structural and cognitive clinical findings. These results underscore the essential role of RAD21 in eukaryotes and emphasize the need for further understanding of the role of cohesin in human development.

Isolated NIBPL missense mutations that cause Cornelia de Lange syndrome alter MAU2 interaction.

Cornelia de Lange syndrome (CdLS; or Brachmann-de Lange syndrome) is a dominantly inherited congenital malformation disorder with features that include characteristic facies, cognitive delays, growth retardation and limb anomalies. Mutations in nearly 60% of CdLS patients have been identified in NIPBL, which encodes a regulator of the sister chromatid cohesion complex. NIPBL, also known as delangin, is a homolog of yeast and amphibian Scc2 and C. elegans PQN-85. Although the exact mechanism of NIPBL function in sister chromatid cohesion is unclear, in vivo yeast and C. elegans experiments and in vitro vertebrate cell experiments have demonstrated that NIPBL/Scc2 functionally interacts with the MAU2/Scc4 protein to initiate loading of cohesin onto chromatin. To test the significance of this model in the clinical setting of CdLS, we fine-mapped the NIBPL-MAU2 interaction domain and tested the functional significance of missense mutations and variants in NIPBL and MAU2 identified in these minimal domains in a cohort of patients with CdLS. We demonstrate that specific novel mutations at the N-terminus of the MAU2-interacting domain of NIBPL result in markedly reduced MAU2 binding, although we appreciate no consistent clinical difference in the small group of patients with these mutations. These data suggest that factors in addition to MAU2 are essential in determining the clinical features and severity of CdLS.

The dystonia gene DYT1 is repressed by the transcription factor THAP1 (DYT6).

Mutations in THAP1 have been associated with dystonia 6. THAP1 encodes a transcription factor with mostly unknown targets. We tested the hypothesis that THAP1 regulates the expression of DYT1 (TOR1A), another dystonia-causing gene. After characterization of the TOR1A promoter, we demonstrate that THAP1 binds to the core promoter of TOR1A. Further, we report that wild type THAP1 represses the expression of TOR1A, whereas dystonia 6-associated mutant THAP1 results in decreased repression of TOR1A. Our data demonstrate that THAP1 regulates the transcription of TOR1A, suggesting transcriptional dysregulation as a cause of dystonia.

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3.

Trps1, the gene mutated in human Tricho-Rhino-Phalangeal syndrome, represents an atypical member of the GATA-family of transcription factors. Here we show that Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3 in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types.