Introduction

Temporospatial regulation of gene expression is critical for proper functioning of all cellular processes. At any given time, thousands of genes need to be activated or repressed in a precise and coordinated manner. To achieve this, multiple mechanisms are employed by cells, including: (1) the recruitment of trans- and cis-acting elements; (2) the modification of DNA accessibility by chromatin remodeling complexes and histone modifiers and (3) the recruitment or mobilization of the RNA polymerase II machinery (Tang et al. 2010).

Over recent years, mutations in multiple transcriptional regulators have been linked to a growing spectrum of neurodevelopmental disorders, recently grouped into the emerging category of so-called “transcriptomopathies” (Yuan et al. 2015; Izumi 2016). Patients with these syndromes share a number of clinical features including growth retardation, intellectual disability, developmental delay and a combination of similar facial features. Cornelia de Lange syndrome (CdLS, OMIM #122470, 300590, 610759, 300882, and 614701) is a rare congenital multisystem disorder that falls within this category. To date, mutations in five different cohesin-related genes, NIPBL, SMC1A, SMC3, RAD21 and HDAC8, have been described to cause several subsets of CdLS, but further locus heterogeneity is predicted (Krantz et al. 2004; Tonkin et al. 2004; Musio et al. 2006; Deardorff et al. 2007, 2012a, b). The cohesin complex is well known for its role in mediating sister chromatid cohesion and DNA double-strand break repair (Mehta et al. 2013). However, in the last decade, extensive analysis of the function and structure of cohesin has revealed that the complex is involved in many other chromatin-related processes, which include transcriptional regulation, chromatin remodeling, chromosome condensation and morphogenesis (Mehta et al. 2013). Consistent with these other key functions, cell lines from patients with CdLS do not display sister chromatid cohesion defects (Castronovo et al. 2009). This underscores that the etiopathology of CdLS and related disorders is not directly linked to the disruption of sister chromatid cohesion but to the altered ability of the cohesin complex to mediate other more dosage-sensitive cellular functions such as transcriptional regulation (Liu and Krantz 2009).

Consistent with a central role of transcriptional dysregulation in patients with CdLS or CdLS-overlapping phenotypes, mutations in chromatin-associated factors other than cohesin have recently been described. These include alterations in the transcriptional regulator genes EP300, AFF4, ANKRD11 and KMT2A, which have been previously associated with other multisystem developmental disorders that are frequently considered in the differential diagnosis of CdLS (Ansari et al. 2014; Woods et al. 2014; Izumi et al. 2015; Yuan et al. 2015; Parenti et al. 2016a). Mutations in EP300, encoding an acetyltransferase, cause Rubinstein–Taybi syndrome type 2 (RSTS2, OMIM #613684), mutations in the transcriptional repressor ANKRD11 lead to KBG syndrome (KBGS, OMIM #148050) and mutations in the histone methyltransferase KMT2A cause Wiedemann–Steiner syndrome (WDSTS, OMIM #605130). Very recently, alterations of AFF4, encoding a core component of the super elongation complex, have been described as the cause of a new CdLS-overlapping syndrome (CHOPS, OMIM #616368) (Roelfsema et al. 2005; Sirmaci et al. 2011; Jones et al. 2012; Izumi et al. 2015). Notably, all of these disorders demonstrate a significant clinical overlap with CdLS, each presenting with shared clinical features that include growth retardation, intellectual disability, developmental delay and facial dysmorphisms such as synophrys, a short nose with anteverted nares, a smooth philtrum and a thin upper lip. In addition to the identification of mutations in transcriptional regulatory genes that can lead to a CdLS-overlapping phenotype, mutations in “CdLS” genes can underlie clinical phenotypes attributed to mutations in other chromatin regulators. For example, Coffin–Siris syndrome (CSS, OMIM #135900) and Nicolaides–Baraitser syndrome (NCBRS, OMIM #601358), which themselves demonstrate some clinical overlap with CdLS, are caused by mutations in different subunits of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, that shows some shared functions with cohesin (Santen et al. 2012; Tsurusaki et al. 2012). Further strengthening this connection, a direct functional interaction between the cohesin loader NIPBL and the SWI/SNF complex in yeast has been recently demonstrated. Particularly, Scc2, the yeast orthologue of NIPBL, is recruited by the SWI/SNF complex to broad nucleosome-free regions, which both subsequently help to maintain (Lopez-Serra et al. 2014). These findings directly indicate the combined roles of cohesin and SWI/SNF in regulating the expression of common target genes and/or genomic regulatory elements.

To further elucidate this important interaction in human development, here, we report on the clinical and molecular characterization of seven patients presenting with features overlapping CdLS, for whom the analysis of the five known CdLS-genes did not identify any disease-causing mutation. Subsequent use of multiple next-generation sequencing technologies identified mutations in key chromatin-associated factors genetically different from cohesin. Mutations in the methyltransferases KMT2A or SETD5 were identified in three patients, and two different subunits of the chromatin-remodeler SWI/SNF complex were mutated in four. To complement this, eight unsolved patients with a clinical diagnosis of CSS/NCBRS were investigated for the presence of mutations in the known CdLS genes. This led to the identification of a missense substitution in NIPBL in one patient with CSS, who was previously described by Wieczorek and colleagues in 2013, attesting that interrelated genes underpin closely similar phenotypes.

In summary, our findings suggest that mutations in different but functionally related chromatin-associated factors might result in strongly overlapping clinical pictures. As the primary role of these factors concerns chromatin, and only indirectly regulation of transcription, we propose that these conditions could be grouped within the category of “chromatin dysregulation disorders” rather than “transcriptomopathies” per se. Also, these findings underscore the necessity to further identify and molecularly characterize the disease-relevant mechanisms involved in the regulation of chromatin function both in normal and pathological backgrounds. Not only will this be of utmost importance for making accurate clinical and molecular diagnoses, but also will it be critical in understanding the pathogenesis and developing therapies that may serve to improve a wider range of disorders caused by disruptions in chromatin regulation.

Materials and methods

Patients

All patients were recruited as part of an international collaboration between investigators in Germany, Austria, Spain, Turkey and Colombia. The study was performed according to the Declaration of Helsinki protocols and was approved by the local institutional review board (ethical votum 12-5089-BO for CRANIRARE and 08-3663 forMRNET). Informed consent was obtained from all individuals and/or their legal guardians included in this study. An additional consent was obtained for the publication of photographic material identifying patients. All children were born to healthy parents and underwent complete physical and dysmorphology evaluation by their referring clinical geneticists. A list of clinical features observed in each patient is provided in Table 1.

Table 1 Clinical features of the reported patients
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DNA isolation

Genomic DNA was isolated from blood or buccal mucosa. Extraction of the genomic material was performed with the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) or with the Gentra Puregene Buccal Cell Kit (Qiagen) according to the manufacturer’s instruction.

Targeted next-generation sequencing

Targeted next-generation sequencing was based on the design of a custom-made gene panel for the Personal Genome Machine (Ion Torrent PGM) with the AmpliSeq Designer Tool (Life Technologies, Darmstadt, Germany) as previously described (Braunholz et al. 2015).

Exome sequencing

Targeted enrichment was performed using the SureSelect XT Human All Exon 50 Mb kit, versions 4 or 5 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on HiSeq 2500 systems (Illumina, San Diego, CA, USA). Read tags were aligned with the human reference genome hg19 (GRCh37) using Burrows—Wheeler Aligner version 0.7.5 (Li and Durbin 2010). Variant calling was performed using SAMtools version 0.1.18, PINDEL version 0.2.4t and ExomeDepth version 1.0.0. Subsequently, variants were filtered using the SAMtools varFilter script. Shortly, variants that were predicted to impact the coding sequences and with a frequency in the population lower than 2% were retained. Resulting variants were filtered according to a dominant/de novo inheritance model.

Sanger sequencing

PCR products were sequenced with the Big Dye® terminator v3.1 Sequencing Kit and run on the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Electropherograms were analyzed with ChromasPro Software version 1.7.6 (Technelysium Pty Ltd., Tewantin QLD, Australia) and aligned with the wild-type sequences of the respective genes. Electropherograms of patients with point mutations are available as Online Resource 1.

CGH-array

Genomic DNA was tested with the qChip Post Oligonucleotide microarray (Quantitative Genomic Medicine Laboratories, Barcelona, Spain). This microarray comprises 60,000 probes mainly located in pericentromeric and subtelomeric regions, as well as regions associated to syndromes caused by recurrent genomic alterations. The average median probes spacing is 35 Kb, and the resolution is of 100–125 Kb. After hybridization with DNA from patient and reference samples, slides were scanned and analyzed for relative gain or loss of fluorescent signals. Genomic region analyses were performed according to the human reference sequence build 37.1 (hg19) with the Genomic Workbench software.

cDNA characterization

RNA of patient 6 was isolated from blood with the PAXgene Blood RNA Kit (Qiagen) following the manufacturer’s protocol. The RNA was subsequently reversely transcribed with random primers using the SuperScript® One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. The cDNA was amplified with the forward primer 5′-CCACAAGGTAACTACTCCAG-3 and the reverse primer 5′-CTGCTTCCAGGCACTTTTCTC-3′. The forward primer spans the junction between exon 8 and 9 of ARID1B, whereas the reverse primer was designed on the junction between exon 14 and 15. Polymerase chain reaction (PCR) was performed with TaqPolymerase (MP Biomedicals, Santa Ana, CA, USA) with an annealing temperature of 58 °C and 90 s elongation at 72 °C. Gel extraction was performed on the resulting 1331-bp band with the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instruction. The purified fragment was ligated with the shuttle vector pGEM®-T Easy (Promega, Madison, WI, USA) by the use of the T4 DNA Ligase (Promega). E. coli DH5α cells were then chemically transformed with the recombinant DNA construct and plated onto LB agar plates containing 100 μg/ml of ampicillin. Transformed bacteria containing the plasmid of interest were grown overnight at 37 °C at 225 rpm in 3 ml of LB medium supplemented with 3 µl of ampicillin. Plasmids were then extracted using the QIAGEN Plasmid Mini Kit (Qiagen) following the manufacturer’s instructions. Sanger sequencing was subsequently performed on plasmids extracted from multiple clones.

Results

Mutations in the methyltransferases KMT2A and SETD5 in patients with CdLS-overlapping phenotypes

Patient 1 is a 15-year-old Austrian male with Turkish ancestry (Fig. 1a–c), who received a clinical diagnosis of CdLS during early childhood. At the age of eight, he was evaluated for growth retardation, mild intellectual disability and was noted to have a low anterior temporal hairline, arched eyebrows with synophrys, long eyelashes, ptosis, bulbous nasal tip and thin upper vermillion border of the lip. Minor limb anomalies included small hands, clinodactyly of the fifth finger and syndactyly of toes. The facial features became more pronounced over time, and at the age of 12 years, hairy elbows were also observed. He had an early puberty at the age of 10 years, obstipation and behavioral problems including nocturnal enuresis and encopresis. Targeted gene panel revealed a de novo nonsense mutation in the KMT2A gene (NM_001197104) on the blood DNA of the patient. The variant, namely c.8590C > T, p.(Gln2864*), falls within exon 27 and is responsible for the introduction of a premature stop codon. Loss-of-function mutations in the histone methyltransferase KMT2A have been so far described in association with Wiedemann–Steiner Syndrome (WDSTS), in a single patient with CdLS-overlapping features and in a single patient with CSS (Jones et al. 2012; Bramswig et al. 2015; Yuan et al. 2015). In combination with the molecular finding, clinical re-evaluation of our patient at the age of 15 demonstrated features consistent with the diagnosis of WDSTS.

Fig. 1
figure 1

Phenotype of the patients of our cohort. a–g Facial appearance of five out of the seven patients with CdLS-overlapping features found to carry mutations in epigenetic genes (writers and remodelers). h Facial appearance of the patient clinically diagnosed as Coffin–Siris who turned out to harbor a mutation in NIPBL

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Patient 2 is a 7-year-old German male of Turkish ancestry (Fig. 1d). Clinical features included mild developmental delay, intellectual disability, a low anterior hairline, thick arched eyebrows with synophrys, long eyelashes, a smooth philtrum and thin upper vermillion border of the lip. Taken together, these features suggested a possible clinical diagnosis of a mild form of CdLS. A de novo frameshift deletion in SETD5 (NM_001080517) was detected on the blood DNA of the patient through whole-exome sequencing. The mutation results in the deletion of two nucleotides in exon 16 (c.2212_2213delAT; p.(Met738Valfs*27)) and is predicted to result in a premature stop codon after 27 amino acids. Clinical re-evaluation of the patient after the molecular finding was still compatible with a mild variant of CdLS.

Consistent with haploinsufficiency of SETD5 causing CdLS-overlapping features, an intragenic deletion involving SETD5 was identified in Patient 3 by array-CGH. A 54-kb-spanning deletion (chr3: 9, 457, 143–9, 511, 190, hg19) extends from introns 2 to 19 and results in the loss of the first 16 protein-coding exons. This patient is an 8-year-old German female with Indian ancestry, diagnosed with a CdLS-overlapping phenotype during early childhood. Her clinical features included severely delayed speech and motor development, cognitive impairment, behavioral problems, feeding problems and facial dysmorphisms that included thick eyebrows, long eyelashes, a depressed nasal bridge, broad nasal tip, smooth philtrum and thin upper vermillion border of the lip. The clinical overlap of this patient’s features with CdLS was confirmed at a recent evaluation. Loss of the methyltransferase-encoding gene SETD5 is thought to be central in causing the 3p25 microdeletion syndrome, characterized by intellectual disability, short stature, microcephaly, hypotonia and heart defects (Kuechler et al. 2015); in addition, loss-of-function mutations of SETD5 have been associated with syndromic and non-specific intellectual disability (Grozeva et al. 2014; Szczaluba et al. 2016). Herein, our data suggest, for the first time, a role of SETD5 in the etiology of CdLS-overlapping features.

Mutations in both cohesin and SWI/SNF complex members result in overlapping clinical phenotypes

Four patients of our CdLS-overlapping cohort were found to carry mutations in subunits of the SWI/SNF chromatin-remodeler complex, a cellular mechanism linked to the neurodevelopmental disorders CSS and NCBRS.

Patient 4 is a 26-year-old German female who presented with marked pre- and post-natal growth retardation, microcephaly, mild developmental delay, intellectual disability, visual and hearing problems, cardiac and gastrointestinal malformations and minor limb anomalies, including proximally set thumbs, clinodactyly of the fifth finger, syndactyly of toes and hypoplastic nails. Her facial dysmorphisms led to the clinical diagnosis of CdLS. These included a low anterior hairline, thick arched eyebrows, synophrys, depressed nasal bridge, long philtrum and thin vermillion border of the lips. A targeted gene panel revealed the presence of a missense substitution in SMARCB1 (NM_001317946), namely c.998A > G, p.(Lys333Arg). Similar to the other four missense substitutions in SMARCB1 previously described in CSS/NCBRS, this mutation falls within the functional SNF5 domain at the C-terminal of the protein (Santen et al. 2012; Tsurusaki et al. 2012; Wieczorek et al. 2013). In addition, the mutation was indicated as disease-causing by the bioinformatic prediction tool MutationTaster (http://www.mutationtaster.org/). Despite the presence of hypoplastic nails, clinical re-evaluation of the patient after the molecular diagnosis demonstrated again that her features were more consistent with CdLS than CSS.

Patient 5 (Fig. 1e) is a 2-year-old female from Colombia presenting with CdLS-overlapping features that included post-natal growth retardation, intellectual disability, delayed speech development, anomalies of the cardiac and central nervous systems, visual problems, hypertrichosis, small hands, proximally set thumbs, clinodactyly of the fifth finger and multiple craniofacial findings including brachycephaly, low anterior hairline, thick and arched eyebrows with synophrys, long eyelashes, depressed nasal bridge, anteverted nostrils, long philtrum, thin upper vermillion border of the lip and thick and everted lower lip. Exome sequencing identified a de novo nonsense mutation in ARID1B (NM_020732), c.2692C > T, p.(Arg898*), previously reported in a patient with a clinical diagnosis of CSS (Wieczorek et al. 2013). Despite a recent clinical re-evaluation that revealed the existence of phenotypical features observed in patients with CdLS and CSS, no clear clinical diagnosis could be assessed for this patient.

Patient 6 is a 7-year-old male of German ancestry (Fig. 1f). In addition to several facial features characteristic for CdLS such as brachycephaly, low anterior hairline, arched eyebrows, broad nasal tip, smooth philtrum and a thin upper lip, the patient presented with short stature, intellectual disability, behavioral problems and developmental delay. Targeted gene panel identified a de novo splicing variant in ARID1B (NM_020732), namely c.3136-2A > G. Subsequent analysis of the cDNA of the patient demonstrated two aberrantly spliced transcripts (Fig. 2), one characterized by the deletion of the first three nucleotides of exon 12, resulting in an in-frame deletion of one amino acid (p.(Lys1046del)). The second transcript lacks the first eight nucleotides of exon 12, resulting in an out-of-frame deletion (p.(Lys1046Leufs*18)). Clinical re-evaluation of the patient after the molecular finding confirmed the better fit with a diagnosis of atypical CdLS than CSS.

Fig. 2
figure 2

cDNA analysis for the characterization of the splicing variant of patient 6 (ARID1B, c.3136-2A > G). Forward and reverse primers were designed on the junctions between exons 8 and 9 and exons 14 and 15, respectively. The expected wild-type product consisted of 1331 bp. Following amplification, a single 1331-bp band was detected in both control cDNA and patient cDNA. The PCR product of the patient underwent gel extraction and subsequent cloning in the pGEM®-T Easy Vector System. After transformation of DH5α strains of E. coli, sequencing analysis of different clones revealed the existence of three distinct splicing isoforms in the patient. a Electropherograms of the three different transcripts of patient 6. Besides the wild-type isoform, two aberrantly spliced transcripts were identified, characterized by a different start site in exon 12. b Graphical representation of the splicing isoforms of patient 3. Capital letters were used to indicate nucleotides of exons 11 and 12, respectively. Small letters were used to indicate the donor and the acceptor sites. The wild-type splicing is indicated by the continuous green line. The dotted red lines indicate, instead, the two aberrantly spliced transcripts. The first transcript is characterized by the deletion of the first three nucleotides of exon 12, thus resulting in an in-frame deletion of one amino acid (p.(Lys1046del)); rather, the second transcript lacks the first eight nucleotides of exon 12, resulting in the out-of-frame deletion p.(Lys1046Leufs*18)

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Patient 7 is a 13-year-old male from Spain (Fig. 1g), who received a clinical diagnosis of CdLS during early childhood. He presented with pre- and post-natal growth retardation, cognitive impairment, mild developmental delay, behavioral problems, cardiac and genitourinary malformations, limb anomalies (brachydactyly, proximally set thumbs, clinodactyly of the fifth finger and hypoplastic nails), feeding and hearing problems, microcephaly and facial features that included a low anterior hairline, thick eyebrows with synophrys, long eyelashes, anteverted nares, smooth philtrum, thin upper vermillion border of the lip and thick lower lip. By array-CGH, he was found to carry a de novo interstitial deletion on chromosome 6 of 4.1 Mb (chr6: 157, 336, 025–161, 564, 564, hg19) that comprised 34 RefSeq genes including ARID1B. A similar deletion containing ARID1B, ZDHHC14, SNX9, SYNJ2, SERAC1, GTF2H5, and TULP4 was previously described in a patient with a clinical diagnosis of CSS (Wieczorek et al. 2013). Interstitial deletions at 6q25 are responsible for a microdeletion syndrome characterized by cognitive impairment, developmental delay, dysmorphic features, structural anomalies of the brain and hearing loss. The critical region for the interstitial 6q microdeletion phenotype was initially mapped to 6q24–6q25 and then further restricted to a 6q25.3 region containing only the protein-coding genes ARID1B and ZDHHC14 (Eash et al. 2005; Nagamani et al. 2009; Michelson et al. 2012). Very recently, haploinsufficiency of ARID1B has been recognized as the main mechanism responsible for the phenotypical manifestations of the 6q25 microdeletion syndrome (Ronzoni et al. 2016). Clinical re-evaluation of our patient after the molecular diagnosis indicated a likely diagnosis of CSS.

The clinical overlap observed between CdLS and CSS/NCBRS, along with the identification of mutations in SWI/SNF subunits in patients with CdLS-overlapping features, prompted us to postulate that patients with a likely diagnosis of CSS/NCBRS might accordingly carry mutations in cohesin proteins. To test this hypothesis, eight CSS/NCBRS patients with no previous molecular diagnosis after SWI/SNF and array screening were analyzed by a targeted gene panel that included all known CdLS genes. By this, we identified a missense substitution in NIPBL in one German female of Turkish ancestry who was diagnosed as CSS (Patient 8, Fig. 1h) and was previously described (patient K2633 in Wieczorek et al. 2013). The mutation c.6886A > G; p.(Ser2296Gly), that falls in exon 40 of NIPBL, alters a region frequently mutated in patients with CdLS (Gillis et al. 2004; Selicorni et al. 2007; Pié et al. 2010) and was predicted as disease-causing by the bioinformatic prediction tool MutationTaster. However, the clinical features of this patient are quite consistent with the initial diagnosis of CSS and include coarse facies with frontal bossing, thick eyebrows, a broad nasal tip with anteverted nostrils, a wide mouth with a thin upper vermillion border of the lip and a thick and everted lower lip and hypoplastic nails.

Discussion

In this work, we demonstrate a remarkable and consistent overlap of molecular and clinical findings of individuals with mutations in cohesin, SWI/SNF complex members and methyltransferases. This observation was developed from the analysis of patients with a tentative diagnosis of CdLS, a neurodevelopmental multisystem disorder characterized by specific facial features and a wide range of overlapping phenotypes (Izumi 2016). Mutations in genes encoding either subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex have been shown to cause different subentities of the broad CdLS spectrum (Krantz et al. 2004; Tonkin et al. 2004; Musio et al. 2006; Deardorff et al. 2007, 2012a, b). The wide-ranging effects of cohesin mutations are not surprising, as cohesin is a highly conserved protein complex involved in multiple aspects of chromatin biology, including sister chromatid cohesion, transcriptional regulation, long-distance genomic interactions, chromatin-remodeling, DNA damage response and chromosome condensation (Mehta et al. 2013). Mutations in NIPBL account for more than half of the patients with CdLS (Gillis et al. 2004; Selicorni et al. 2007; Pié et al. 2010), and these individuals tend to present with a more typical facial appearance and limb deficiencies. These patients can have a phenotype that encompasses a range of mild to severe developmental and cognitive delay, often with absence of speech, and severe pre- and post-natal growth retardation (Selicorni et al. 2007). Mutations in the remaining four genes are found in about 10% of the patients with CdLS and are associated with fewer major structural malformations and less growth retardation, although their intellectual disability can range from mild to profound (Deardorff et al. 2012a, b; Gervasini et al. 2013; Kaiser et al. 2014; Gil-Rodríguez et al. 2015; Parenti et al. 2016b). These latter patients frequently demonstrate clinical features that overlap those of patients carrying mutations in other chromatin-associated proteins. Accordingly, mutations in various chromatin-associated factors aside from cohesin, have been identified in patients with CdLS or CdLS-overlapping phenotypes. These genes include EP300, AFF4, ANKRD11 and KMT2A (Ansari et al. 2014; Woods et al. 2014; Izumi et al. 2015; Yuan et al. 2015; Parenti et al. 2016a). Notably, mutations in all of these genes have previously been reported in association with other multisystem developmental disorders that are frequently included in the differential diagnosis for CdLS (Roelfsema et al. 2005; Sirmaci et al. 2011; Jones et al. 2012; Izumi et al. 2015). Correspondingly, in our cohort of patients with features resembling CdLS, we identified one patient with a nonsense mutation in the methyltransferase-encoding gene KMT2A. Currently, mutations in this gene have been described in association with WDSTS (Jones et al. 2012). Also, Yuan and colleagues recently identified a mutation in this gene in a patient with features consistent with a diagnosis of CdLS, whereas another study identified a mutation in KMT2A in a patient with a tentative diagnosis of CSS (Bramswig et al. 2015; Yuan et al. 2015). Our patient shares with these previously published patients a number of clinical features including short stature, intellectual disability, hypertrichosis, fifth finger clinodactyly, long eyelashes, long philtrum and thin upper lip. However, our patient also demonstrates the presence of hairy elbows, a feature typical for WDSTS that was not observed in the patients described in 2015 (Bramswig et al. 2015; Yuan et al. 2015).

In addition to KMT2A, we have also identified loss-of-function mutations in the chromatin regulatory methyltransferase gene SETD5 in two patients of our cohort. Haploinsufficiency of SETD5 has been shown to underlie the 3p25 microdeletion syndrome and loss-of-function mutations of this gene have also been shown to cause syndromic and non-specific intellectual disability (Grozeva et al. 2014; Kuechler et al. 2015; Szczaluba et al. 2016). The phenotypic features of our patients with mutations in SETD5 share similarities with the previously described patients, including a variable level of cognitive impairment and intellectual disability, ear anomalies, abnormal shape of the nose and long philtrum (Grozeva et al. 2014; Kuechler et al. 2015; Szczaluba et al. 2016). Our findings underscore the importance of methyltransferases in chromatin remodeling and suggest that mutations in these genes might alter chromatin regulation in a similar manner as cohesin, resulting in a spectrum of clinical disorders that displays pronounced overlap.

In addition, for the very first time, we have established a direct clinical link to support the recently unveiled molecular link between CdLS and CSS/NCBRS (Lopez-Serra et al. 2014) through the identification of four patients with CdLS-overlapping features with mutations in SWI/SNF subunits and of one CSS patient with a mutation in NIPBL. We postulate that additional mutations will be identified with the analysis of a larger number of patients. In our cohort, we have observed overlapping features of CdLS and CSS/NCBRS in each individual and, frequently, several unique features typical of each syndrome. For instance, patients 4 and 7 presented with hypoplastic nails, a feature that is frequently described in association with CSS/NCBRS. In contrast, patients 5 and 6 demonstrated brachycephaly, a finding mainly described in CdLS. These findings indicate that the high degree of clinical overlap of all three syndromes may be explained at least partially by the significant overlap of functions between cohesin and SWI/SNF, both involved in transcriptional regulation, chromatin remodeling and epigenetic modifications. This model is further supported by the direct physical interaction identified between these two protein complexes that has been recently detected in yeast, where the SWI/SNF complex recruits the cohesin loader NIPBL onto broad nucleosome-free chromatin regions that NIPBL subsequently helps to maintain in a nucleosome-free transcriptionally active state (Lopez-Serra et al. 2014).

Notably, three out of the eight patients of our cohort are of Turkish ancestry (patients 1, 2 and 8). Clinical features typical for CdLS such as a synophrys and/or hirsutism might be more frequently observed in individuals of this ethnicity. All of the three patients described here do, indeed, present with synophrys. For this reason, a systematic evaluation of the ethnic background is recommendable for the assessment of the most appropriate clinical diagnosis.

In addition, our findings further support previous observations indicating no clear correlation of the genetic variant with the clinical manifestation of the phenotype, possibly due to the broad variability of clinical features seen in patients with CdLS. Therefore, we strongly recommend the application of next-generation sequencing technologies such as exome sequencing or gene panel sequencing including multiple chromatin regulators to allow the most efficient molecular analysis of patients with a clinical diagnosis of CdLS or CdLS-like phenotypes.

In summary, the identification of mutations in a growing number of chromatin-associated regulators in developmental syndromes strongly indicates that shared functions of these distinct gene products act in common cellular mechanisms and pathways. As a consequence, syndromes caused by mutations in these genes are subsequently characterized by significantly overlapping phenotypes that may be difficult to discern from one another. We propose that these disorders could be aptly grouped into the category of “chromatin dysregulation disorders”. The identification of the molecular mechanisms will be of utmost importance for making accurate clinical and molecular diagnoses. Furthermore, understanding the pathogenesis will be essential to develop treatments for individuals with a wide range of clinical features caused by disruption of chromatin regulation.