Diagnosis and management in Rubinstein-Taybi syndrome: first international consensus statement.

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.

ARF1-related disorder: phenotypic and molecular spectrum.

PURPOSE: ARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder. METHODS: We collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated. RESULTS: De novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder. CONCLUSION: We confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.

Autosomal Dominant MPAN: Mosaicism Expands the Clinical Spectrum to Atypical Late-Onset Phenotypes.

BACKGROUND: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is caused by mutations in the C19orf12 gene. MPAN typically appears in the first two decades of life and presents with progressive dystonia-parkinsonism, lower motor neuron signs, optic atrophy, and abnormal iron deposits predominantly in the basal ganglia. MPAN, initially considered as a strictly autosomal recessive disease (AR), turned out to be also dominantly inherited (AD). OBJECTIVES: Our aim was to better characterize the clinical, molecular, and functional spectra associated with such dominant pathogenic heterozygous C19orf12 variants. METHODS: We collected clinical, imaging, and molecular information of eight individuals from four AD-MPAN families and obtained brain neuropathology results for one. Functional studies, focused on energy and iron metabolism, were conducted on fibroblasts from AD-MPAN patients, AR-MPAN patients, and controls. RESULTS: We identified four heterozygous C19orf12 variants in eight AD-MPAN patients. Two of them carrying the familial variant in mosaic displayed an atypical late-onset phenotype. Fibroblasts from AD-MPAN showed more severe alterations of iron storage metabolism and autophagy compared to AR-MPAN cells. CONCLUSION: Our data add strong evidence of the realness of AD-MPAN with identification of novel monoallelic C19orf12 variants, including at the mosaic state. This has implications in diagnosis procedures. We also expand the phenotypic spectrum of MPAN to late onset atypical presentations. Finally, we demonstrate for the first time more drastic abnormalities of iron metabolism and autophagy in AD-MPAN than in AR-MPAN. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Deep intronic NIPBL de novo mutations and differential diagnoses revealed by whole genome and RNA sequencing in Cornelia de Lange syndrome patients.

Cornelia de Lange syndrome (CdLS; MIM# 122470) is a rare developmental disorder. Pathogenic variants in 5 genes explain approximately 50% cases, leaving the other 50% unsolved. We performed whole genome sequencing (WGS) ± RNA sequencing (RNA-seq) in 5 unsolved trios fulfilling the following criteria: (i) clinical diagnosis of classic CdLS, (ii) negative gene panel sequencing from blood and saliva-isolated DNA, (iii) unaffected parents' DNA samples available and (iv) proband's blood-isolated RNA available. A pathogenic de novo mutation (DNM) was observed in a CdLS differential diagnosis gene in 3/5 patients, namely POU3F3, SPEN, and TAF1. In the other two, we identified two distinct deep intronic DNM in NIPBL predicted to create a novel splice site. RT-PCRs and RNA-Seq showed aberrant transcripts leading to the creation of a novel frameshift exon. Our findings suggest the relevance of WGS in unsolved suspected CdLS cases and that deep intronic variants may account for a proportion of them.

Clinical Utility of a Unique Genome-Wide DNA Methylation Signature for KMT2A-Related Syndrome.

Wiedemann-Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic variants in the KMT2A gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare KMT2A variants can be challenging. A genome-wide DNA methylation episignature for KMT2A-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the KMT2A gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from KMT2A mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.

Triple diagnosis of Wiedemann-Steiner, Waardenburg and DLG3-related intellectual disability association found by WES: A case report.

BACKGROUND: The development of whole-exome sequencing (WES) and whole-genome sequencing (WGS) for clinical purposes now allows the identification of multiple pathogenic variants in patients with a rare disease. This occurs even when a single causative gene was initially suspected. We report the case of an 8-year-old patient with global developmental delays and dysmorphic features, with a possibly pathogenic variant in three distinct genes. METHODS: Trio-based exome sequencing was performed by IntegraGen SA (Evry, France), on an Illumina HiSeq4000 (Illumina, San Diego, CA, USA). Sanger sequencing was performed to confirm the variants that were found. RESULTS: WES showed the presence of three possibly deleterious variants: KMT2A: c.9068delA;p.Gln3023Argfs*3 de novo, PAX3: c.530C>G;p.Ala177Gly de novo and DLG3: c.127delG;p.Asp43Metfs*22 hemizygous inherited from the mother. KMT2A pathogenic variants are involved in Wiedemann-Steiner syndrome, and PAX3 is the gene responsible for Waardenburg syndrome. DLG3 variants have been described in a non-syndromic X-related intellectual disability. CONCLUSIONS: Considering the dysmorphic features and intellectual disability presented by this patient, these three variants were imputed as pathogenic and their association was considered responsible for his phenotype. Dual molecular diagnoses have already been found by WES in several cohorts with an average of diagnostic yield of 7%. This case demonstrates and reminds us of the importance of analyzing exomes rigorously and exhaustively because, in some cases (< 10%), it can explain superimposed traits or blended phenotypes.

Fetal phenotype of Rubinstein-Taybi syndrome caused by CREBBP mutations.

Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is an autosomal dominant developmental disorder characterized by facial dysmorphism, broad thumbs and halluces associated with intellectual disability. RSTS is caused by alterations in CREBBP (about 60%) and EP300 genes (8%). RSTS is often diagnosed at birth or during early childhood but generally not suspected during antenatal period. We report nine cases of well-documented fetal RSTS. Two cases were examined after death in utero at 18 and 35 weeks of gestation and seven cases after identification of ultrasound abnormalities and termination of pregnancy. On prenatal sonography, a large gallbladder was detected in two cases, and brain malformations were noted in four cases, especially cerebellar hypoplasia. However, the diagnosis of RSTS has not been suggested during pregnancy. Fetal autopsy showed that all fetuses had large thumbs and/or suggestive facial dysmorphism. A CREBBP gene anomaly was identified in all cases. Alterations were similar to those found in typical RSTS children. This report will contribute to a better knowledge of the fetal phenotype to consider the hypothesis of RSTS during pregnancy. Genotyping allows reassuring genetic counseling.

PADDAS syndrome associated with hair dysplasia caused by a de novo missense variant of PUM1.

PUM1 has been very recently reported as responsible for a new form of developmental disorder named PADDAS syndrome. We describe here an additional patient with early onset developmental delay, epilepsy, microcephaly, and hair dysplasia, with a de novo heterozygous missense variant of PUM1: c.3439C > T, p.(Arg1147Trp). This variant was absent from databases and predicted deleterious by multiple softwares. The same missense variant has been reported by Gennarino et al., in a girl with much more severe epilepsy. Our report is in favor of a variable expressivity of PADDAS syndrome, and broadens the phenotypic spectrum with the description of hair dysplasia.

Gain-of-Function Mutation in Filamin A Potentiates Platelet Integrin αIIbß3 Activation.

OBJECTIVE: Dominant mutations of the X-linked filamin A (FLNA) gene are responsible for filaminopathies A, which are rare disorders including brain periventricular nodular heterotopia, congenital intestinal pseudo-obstruction, cardiac valves or skeleton malformations, and often macrothrombocytopenia. APPROACH AND RESULTS: We studied a male patient with periventricular nodular heterotopia and congenital intestinal pseudo-obstruction, his unique X-linked FLNA allele carrying a stop codon mutation resulting in a 100-amino acid-long FLNa C-terminal extension (NP_001447.2: p.Ter2648SerextTer101). Platelet counts were normal, with few enlarged platelets. FLNa was detectable in all platelets but at 30% of control levels. Surprisingly, all platelet functions were significantly upregulated, including platelet aggregation and secretion, as induced by ADP, collagen, or von Willebrand factor in the presence of ristocetin, as well as thrombus formation in blood flow on a collagen or on a von Willebrand factor matrix. Most importantly, patient platelets stimulated with ADP exhibited a marked increase in αIIbß3 integrin activation and a parallel increase in talin recruitment to ß3, contrasting with normal Rap1 activation. These results are consistent with the mutant FLNa affecting the last step of αIIbß3 activation. Overexpression of mutant FLNa in the HEL megakaryocytic cell line correlated with an increase (compared with wild-type FLNa) in PMA-induced fibrinogen binding to and in talin and kindlin-3 recruitment by αIIbß3. CONCLUSIONS: Altogether, our results are consistent with a less binding of mutant FLNa to ß3 and the facilitated recruitment of talin by ß3 on platelet stimulation, explaining the increased αIIbß3 activation and the ensuing gain-of-platelet functions.

CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.

Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles.

Otopalatodigital spectrum disorders: refinement of the phenotypic and mutational spectrum.

Otopalatodigital spectrum disorders (OPDSD) constitute a group of dominant X-linked osteochondrodysplasias including four syndromes: otopalatodigital syndromes type 1 and type 2 (OPD1 and OPD2), frontometaphyseal dysplasia, and Melnick-Needles syndrome. These syndromes variably associate specific facial and extremities features, hearing loss, cleft palate, skeletal dysplasia and several malformations, and show important clinical overlap over the different entities. FLNA gain-of-function mutations were identified in these conditions. FLNA encodes filamin A, a scaffolding actin-binding protein. Here, we report phenotypic descriptions and molecular results of FLNA analysis in a large series of 27 probands hypothesized to be affected by OPDSD. We identified 11 different missense mutations in 15 unrelated probands (n=15/27, 56%), of which seven were novel, including one of unknown significance. Segregation analyses within families made possible investigating 20 additional relatives carrying a mutation. This series allows refining the phenotypic and mutational spectrum of FLNA mutations causing OPDSD, and providing suggestions to avoid the overdiagnosis of OPD1.

CREBBP mutations in individuals without Rubinstein-Taybi syndrome phenotype.

Mutations in CREBBP cause Rubinstein-Taybi syndrome. By using exome sequencing, and by using Sanger in one patient, CREBBP mutations were detected in 11 patients who did not, or only in a very limited manner, resemble Rubinstein-Taybi syndrome. The combined facial signs typical for Rubinstein-Taybi syndrome were absent, none had broad thumbs, and three had only somewhat broad halluces. All had apparent developmental delay (being the reason for molecular analysis); five had short stature and seven had microcephaly. The facial characteristics were variable; main characteristics were short palpebral fissures, telecanthi, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Six patients had autistic behavior, and two had self-injurious behavior. Other symptoms were recurrent upper airway infections (n = 5), feeding problems (n = 7) and impaired hearing (n = 7). Major malformations occurred infrequently. All patients had a de novo missense mutation in the last part of exon 30 or beginning of exon 31 of CREBBP, between base pairs 5,128 and 5,614 (codons 1,710 and 1,872). No missense or truncating mutations in this region have been described to be associated with the classical Rubinstein-Taybi syndrome phenotype. No functional studies have (yet) been performed, but we hypothesize that the mutations disturb protein-protein interactions by altering zinc finger function. We conclude that patients with missense mutations in this specific CREBBP region show a phenotype that differs substantially from that in patients with Rubinstein-Taybi syndrome, and may prove to constitute one (or more) separate entities. © 2016 Wiley Periodicals, Inc.

Phenotype and genotype in 52 patients with Rubinstein-Taybi syndrome caused by EP300 mutations.

Rubinstein-Taybi syndrome (RSTS) is a developmental disorder characterized by a typical face and distal limbs abnormalities, intellectual disability, and a vast number of other features. Two genes are known to cause RSTS, CREBBP in 60% and EP300 in 8-10% of clinically diagnosed cases. Both paralogs act in chromatin remodeling and encode for transcriptional co-activators interacting with >400 proteins. Up to now 26 individuals with an EP300 mutation have been published. Here, we describe the phenotype and genotype of 42 unpublished RSTS patients carrying EP300 mutations and intragenic deletions and offer an update on another 10 patients. We compare the data to 308 individuals with CREBBP mutations. We demonstrate that EP300 mutations cause a phenotype that typically resembles the classical RSTS phenotype due to CREBBP mutations to a great extent, although most facial signs are less marked with the exception of a low-hanging columella. The limb anomalies are more similar to those in CREBBP mutated individuals except for angulation of thumbs and halluces which is very uncommon in EP300 mutated individuals. The intellectual disability is variable but typically less marked whereas the microcephaly is more common. All types of mutations occur but truncating mutations and small rearrangements are most common (86%). Missense mutations in the HAT domain are associated with a classical RSTS phenotype but otherwise no genotype-phenotype correlation is detected. Pre-eclampsia occurs in 12/52 mothers of EP300 mutated individuals versus in 2/59 mothers of CREBBP mutated individuals, making pregnancy with an EP300 mutated fetus the strongest known predictor for pre-eclampsia. © 2016 Wiley Periodicals, Inc.

Heterogeneity of platelet functional alterations in patients with filamin A mutations.

OBJECTIVE: We examined platelet functions in 4 unrelated patients with filaminopathy A caused by dominant mutations of the X-linked filamin A (FLNA) gene. METHODS AND RESULTS: Patients P1, P2, and P4 exhibited periventricular nodular heterotopia, heterozygozity for truncating FLNA mutations, and thrombocytopenia (except P2). P3 exhibited isolated thrombocytopenia and heterozygozity for a p.Glu1803Lys FLNA mutation. Truncated FLNA was undetectable by Western blotting of P1, P2, and P4 platelets, but full-length FLNA was detected at 37%, 82%, and 57% of control, respectively. P3 FLNA (p.Glu1803Lys and full-length) was assessed at 79%. All patients exhibited a platelet subpopulation negative for FLNA. Platelet aggregation, secretion, glycoprotein VI signaling, and thrombus growth on collagen were decreased for P1, P3, and P4, but normal for P2. For the 2 patients analyzed (P1 and P4), spreading was enhanced and, more markedly, in FLNA-negative platelets, suggesting that FLNA negatively regulates cytoskeleton reorganization. Platelet adhesion to von Willebrand factor under flow correlated with platelet full-length FLNA content: markedly reduced for P1 and P4 and unchanged for P2. Interestingly, von Willebrand factor flow adhesion was increased for P3, consistent with a gain-of-function effect enhancing glycoprotein Ib-IX-V/von Willebrand factor interaction. These results are consistent with a positive role for FLNA in platelet adhesion under high shear. CONCLUSIONS: FLNA mutation heterogeneity correlates with different platelet functional impacts and points to opposite regulatory roles of FLNA in spreading and flow adhesion under shear.

Transcriptome and acetylome profiling identify crucial steps of neuronal differentiation in Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RTS) is a rare and severe genetic developmental disorder characterized by multiple congenital anomalies and intellectual disability. CREBBP and EP300, the two genes known to cause RTS encode transcriptional coactivators with a catalytic lysine acetyltransferase (KAT) activity. Loss of CBP or p300 function results in a deficit in protein acetylation, in particular at histones. In RTS, nothing is known on the consequences of the loss of histone acetylation on the transcriptomic profiles during neuronal differentiation. To address this question, we differentiated induced pluripotent stem cells from RTS patients carrying a recurrent CREBBP mutation that inactivates the KAT domain into cortical and pyramidal neurons. By comparing their acetylome and their transcriptome at different neuronal differentiation time points, we identified 25 specific acetylated histone residues altered in RTS. We also identified the transition between neural progenitors and immature neurons as a critical step of the differentiation process, with a delayed neuronal maturation in RTS. Overall, this study opens new perspectives in the definition of epigenetic biomarkers for RTS, whose methodology could be extended to other chromatinopathies.

Prevalence of Fabry disease in patients with chronic pain: Lessons from the DOUFAB and DOUFABIS studies.

BACKGROUND: Fabry disease (FD) is a rare X-linked lysosomal disorder caused by alpha-galactosidase deficiency consecutive to a pathogenic variant in the GLA gene. Age at onset is highly variable, with a wide clinical spectrum including frequent renal, cardiac, skin and nervous system manifestations. Since pain can be an indicator of underlying FD, we wanted to estimate the prevalence of FD in a population of chronic pain patients. METHODS: Two studies, DOUFAB and DOUFABIS, were carried out in expert centers for chronic pain to assess the prevalence of FD by measuring alpha galactosidase A activity in men and analysing the GLA gene in women. RESULTS: Analysis of 893 patients, essentially adults, led to the diagnosis of FD in one female patient, now treated with enzyme replacement therapy. CONCLUSIONS: The prevalence of FD is estimated about 1/1000 in our population of men and women suffering from various chronic pain. This is nearly the prevalence of FD observed in other previously screened high-risk populations with renal failure. SIGNIFICANCE: Although a systematic search for FD does not seem relevant in the context of unexplained chronic pain in adults, a positive family history of FD or the presence of additional FD related organ features must lead to consider this rare disease diagnosis. Therefore, pain specialists need to be aware of main features of FD, including pain characteristics.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles.

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n = 71) or p300 (n = 11) (NP_004371.2 residues 1,705-1,875 and NP_001420.2 residues 1,668-1,833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc-binding residues of two zinc-finger domains (ZZ and TAZ2) and within the first α helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in nine out of 10 tested individuals) and TAZ2 domain in CBP (in 14 out of 20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21 out of 21). Phenotypes including intellectual disability of varying degree and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.