Molecular consequences of PQBP1 deficiency, involved in the X-linked Renpenning syndrome.

Mutations in the PQBP1 gene (polyglutamine-binding protein-1) are responsible for a syndromic X-linked form of neurodevelopmental disorder (XL-NDD) with intellectual disability (ID), named Renpenning syndrome. PQBP1 encodes a protein involved in transcriptional and post-transcriptional regulation of gene expression. To investigate the consequences of PQBP1 loss, we used RNA interference to knock-down (KD) PQBP1 in human neural stem cells (hNSC). We observed a decrease of cell proliferation, as well as the deregulation of the expression of 58 genes, comprising genes encoding proteins associated with neurodegenerative diseases, playing a role in mRNA regulation or involved in innate immunity. We also observed an enrichment of genes involved in other forms of NDD (CELF2, APC2, etc). In particular, we identified an increase of a non-canonical isoform of another XL-NDD gene, UPF3B, an actor of nonsense mRNA mediated decay (NMD). This isoform encodes a shorter protein (UPF3B_S) deprived from the domains binding NMD effectors, however no notable change in NMD was observed after PQBP1-KD in fibroblasts containing a premature termination codon. We showed that short non-canonical and long canonical UPF3B isoforms have different interactomes, suggesting they could play distinct roles. The link between PQBP1 loss and increase of UPF3B_S expression was confirmed in mRNA obtained from patients with pathogenic variants in PQBP1, particularly pronounced for truncating variants and missense variants located in the C-terminal domain. We therefore used it as a molecular marker of Renpenning syndrome, to test the pathogenicity of variants of uncertain clinical significance identified in PQPB1 in individuals with NDD, using patient blood mRNA and HeLa cells expressing wild-type or mutant PQBP1 cDNA. We showed that these different approaches were efficient to prove a functional effect of variants in the C-terminal domain of the protein. In conclusion, our study provided information on the pathological mechanisms involved in Renpenning syndrome, but also allowed the identification of a biomarker of PQBP1 deficiency useful to test variant effect.

Bi-allelic TTI1 variants cause an autosomal-recessive neurodevelopmental disorder with microcephaly.

Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.

Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders.

TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

An HNRNPK-specific DNA methylation signature makes sense of missense variants and expands the phenotypic spectrum of Au-Kline syndrome.

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations.

Alternative splicing of BUD13 determines the severity of a developmental disorder with lipodystrophy and progeroid features.

PURPOSE: In this study we aimed to identify the molecular genetic cause of a progressive multisystem disease with prominent lipodystrophy. METHODS: In total, 5 affected individuals were investigated using exome sequencing. Dermal fibroblasts were characterized using RNA sequencing, proteomics, immunoblotting, immunostaining, and electron microscopy. Subcellular localization and rescue studies were performed. RESULTS: We identified a lipodystrophy phenotype with a typical facial appearance, corneal clouding, achalasia, progressive hearing loss, and variable severity. Although 3 individuals showed stunted growth, intellectual disability, and died within the first decade of life (A1, A2, and A3), 2 are adults with normal intellectual development (A4 and A5). All individuals harbored an identical homozygous nonsense variant affecting the retention and splicing complex component BUD13. The nucleotide substitution caused alternative splicing of BUD13 leading to a stable truncated protein whose expression positively correlated with disease expression and life expectancy. In dermal fibroblasts, we found elevated intron retention, a global reduction of spliceosomal proteins, and nuclei with multiple invaginations, which were more pronounced in A1, A2, and A3. Overexpression of both BUD13 isoforms normalized the nuclear morphology. CONCLUSION: Our results define a hitherto unknown syndrome and show that the alternative splice product converts a loss-of-function into a hypomorphic allele, thereby probably determining the severity of the disease and the survival of affected individuals.

Toward clinical and molecular dissection of frontonasal dysplasia with facial skin polyps: From Pai syndrome to differential diagnosis through a series of 27 patients.

Unique or multiple congenital facial skin polyps are features of several rare syndromes, from the most well-known Pai syndrome (PS), to the less recognized oculoauriculofrontonasal syndrome (OAFNS), encephalocraniocutaneous lipomatosis (ECCL), or Sakoda complex (SC). We set up a research project aiming to identify the molecular bases of PS. We reviewed 27 individuals presenting with a syndromic frontonasal polyp and initially referred for PS. Based on strict clinical classification criteria, we could confirm only nine (33%) typical and two (7%) atypical PS individuals. The remaining ones were either OAFNS (11/27-41%) or presenting with an overlapping syndrome (5/27-19%). Because of the phenotypic overlap between these entities, OAFNS, ECCL, and SC can be either considered as differential diagnosis of PS or part of the same spectrum. Exome and/or genome sequencing from blood DNA in 12 patients and from affected tissue in one patient failed to identify any replication in candidate genes. Taken together, our data suggest that conventional approaches routinely utilized for the identification of molecular etiologies responsible for Mendelian disorders are inconclusive. Future studies on affected tissues and multiomics studies will thus be required in order to address either the contribution of mosaic or noncoding variation in these diseases.

MG132 Induces Progerin Clearance and Improves Disease Phenotypes in HGPS-like Patients' Cells.

Progeroid syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS), are premature and accelerated aging diseases, characterized by clinical features mimicking physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type lamins. This mutation activates a cryptic splice site, leading to the production of a truncated prelamin A, called prelamin A ∆50 or progerin, that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations and are named "HGPS-like" patients. They produce progerin and/or other truncated prelamin A isoforms (∆35 and ∆90). We previously found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patients' cells other than those previously described in classical HGPS. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like or classical HGPS.

Formation of keto-type ceramides in palmoplantar keratoderma based on biallelic KDSR mutations in patients.

Functional skin barrier requires sphingolipid homeostasis; 3-ketodihydrosphingosine reductase or KDSR is a key enzyme of sphingolipid anabolism catalyzing the reduction of 3-ketodihydrosphingosine to sphinganine. Biallelic mutations in the KDSR gene may cause erythrokeratoderma variabilis et progressive-4, later specified as PERIOPTER syndrome, emphasizing a characteristic periorifical and ptychotropic erythrokeratoderma. We report another patient with compound heterozygous mutations in KDSR, born with generalized harlequin ichthyosis, which progressed into palmoplantar keratoderma. To determine whether patient-associated KDSR mutations lead to KDSR substrate accumulation and/or unrecognized sphingolipid downstream products in stratum corneum (SC), we analyzed lipids of this and previously published patients with non-identical biallelic mutations in KDSR. In SC of both patients, we identified 'hitherto' unobserved skin ceramides with an unusual keto-type sphingoid base in lesional and non-lesional areas, which accounted for up to 10% of the measured ceramide species. Furthermore, an overall shorter mean chain length of free and bound sphingoid bases was observed-shorter mean chain length of free sphingoid bases was also observed in lesional psoriasis vulgaris SC, but not generally in lesional atopic dermatitis SC. Formation of keto-type ceramides is probably due to a bottle neck in metabolic flux through KDSR and a bypass by ceramide synthases, which highlights the importance of tight intermediate regulation during sphingolipid anabolism and reveals substrate deprivation as potential therapy.

Systematic Profiling of DNMT3A Variants Reveals Protein Instability Mediated by the DCAF8 E3 Ubiquitin Ligase Adaptor.

Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A (DNMT3A) are the most common driver of this state. DNMT3A variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations are unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, and found that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and acute myeloid leukemia development. We investigated the mechanisms underlying the instability using a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and potential therapeutic significance for patients with hematologic disease. SIGNIFICANCE: DNMT3A has emerged as the most important epigenetic regulator and tumor suppressor in the hematopoietic system. Our study represents a systematic and high-throughput method to characterize the molecular impact of DNMT3A missense mutations and the discovery of a regulated destruction mechanism of DNMT3A offering new prognostic and future therapeutic avenues.See related commentary by Ma and Will, p. 23.This article is highlighted in the In This Issue feature, p. 1.

Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.

Phenotypic spectrum and genomics of undiagnosed arthrogryposis multiplex congenita.

BACKGROUND: Arthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families. METHODS: Several genomic approaches were used including genetic mapping of disease loci in multiplex or consanguineous families, TES then WES. Sanger sequencing was performed to identify or validate variants. RESULTS: We achieved disease gene identification in 52.7% of AMC index patients including nine recently identified genes (CNTNAP1, MAGEL2, ADGRG6, ADCY6, GLDN, LGI4, LMOD3, UNC50 and SCN1A). Moreover, we identified pathogenic variants in ASXL3 and STAC3 expanding the phenotypes associated with these genes. The most frequent cause of AMC was a primary involvement of skeletal muscle (40%) followed by brain (22%). The most frequent mode of inheritance is autosomal recessive (66.3% of patients). In sporadic patients born to non-consanguineous parents (n=60), de novo dominant autosomal or X linked variants were observed in 30 of them (50%). CONCLUSION: New genes recently identified in AMC represent 21% of causing genes in our cohort. A high proportion of de novo variants were observed indicating that this mechanism plays a prominent part in this developmental disease. Our data showed the added value of WES when compared with TES due to the larger clinical spectrum of some disease genes than initially described and the identification of novel genes.

The Largest Germline Heterozygous Deletion Encompassing Potocki-Shaffer and WAGR Syndromes Loci to Date: A Case Report.

Potocki-Schaffer syndrome includes multiple exostoses, parietal foramina, and variable developmental delay/intellectual disability. It is associated with a heterozygous deletion of the 11p12p11.2 region. In some cases, the deletion extends to the WAGR locus (11p13p12). We describe here a 9-month-old girl harboring the largest germline heterozygous deletion characterized so far. Oligohydramnios and parietal foramina were noticed during pregnancy. No patient has been diagnosed before with concomitance of these two syndromes during the prenatal period. Cytogenetic diagnosis was anticipated on basis of clinical and radiological signs. Postnatal conventional karyotype confirmed an interstitial 11p deletion: 46,XX,del(11)(p11.2p15.1). Array-comparative genomic hybridization characterized a 29.6 Mb deletion. Our case illustrates the interest of high-resolution genomic approaches to correlate adequately clinical phenotypes with specific genes in suspected contiguous gene deletion syndromes.

Implementation of fetal clinical exome sequencing: Comparing prospective and retrospective cohorts.

PURPOSE: We compared the diagnostic yield of fetal clinical exome sequencing (fCES) in prospective and retrospective cohorts of pregnancies presenting with anomalies detected using ultrasound. We evaluated factors that led to a higher diagnostic efficiency, such as phenotypic category, clinical characterization, and variant analysis strategy. METHODS: fCES was performed for 303 fetuses (183 ongoing and 120 ended pregnancies, in which chromosomal abnormalities had been excluded) using a trio/duo-based approach and a multistep variant analysis strategy. RESULTS: fCES identified the underlying genetic cause in 13% (24/183) of prospective and 29% (35/120) of retrospective cases. In both cohorts, recessive heterozygous compound genotypes were not rare, and trio and simplex variant analysis strategies were complementary to achieve the highest possible diagnostic rate. Limited prenatal phenotypic information led to interpretation challenges. In 2 prospective cases, in-depth analysis allowed expansion of the spectrum of prenatal presentations for genetic syndromes associated with the SLC17A5 and CHAMP1 genes. CONCLUSION: fCES is diagnostically efficient in fetuses presenting with cerebral, skeletal, urinary, or multiple anomalies. The comparison between the 2 cohorts highlights the importance of providing detailed phenotypic information for better interpretation and prenatal reporting of genetic variants.

Two new cases of interstitial 7q35q36.1 deletion including CNTNAP2 and KMT2C.

BACKGROUND: Terminal deletions of the long arm of chromosome 7 are well known and frequently associated with syndromic holoprosencephaly due to the involvement of the SHH (aliases HHG1, SMMCI, TPT, TPTPS, and MCOPCB5) gene region. However, interstitial deletions including CNTNAP2 (aliases Caspr2, KIAA0868, and NRXN4) and excluding the SHH region are less common. METHODS: We report the clinical and molecular characterization associated with pure 7q35 and 7q35q36.1 deletion in two unrelated patients as detected by oligonucleotide-based array-CGH analysis. RESULTS: The common clinical features were abnormal maternal serum screening during first-trimester pregnancy, low occipitofrontal circumference at birth, hypotonia, abnormal feet, developmental delay, impaired language development, generalized seizures, hyperactive behavior, friendly personality, and cranio-facial dysmorphism. Both deletions occurred de novo and sequencing of CNTNAP2, a candidate gene for epilepsy and autism showed absence of mutation on the contralateral allele. CONCLUSION: Combined haploinsufficiency of GALNTL5 (alias GalNAc-T5L), CUL1, SSPO (aliases SCO-spondin, KIAA0543, and FLJ36112), AOC1 (alias DAO), RHEB, and especially KMT2C (alias KIAA1506 and HALR) with monoallelic disruption of CNTNAP2 may explain neurologic abnormalities, hypotonia, and exostoses. Haploinsufficiency of PRKAG2 (aliases AAKG, AAKG2, H91620p, WPWS, and CMH6) and KCNH2 (aliases Kv11.1, HERG, and erg1) genes may be responsible of long QT syndrome observed for one patient.

Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus.

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.

Touch and olfaction/taste differentiate children carrying a 16p11.2 deletion from children with ASD.

BACKGROUND: Sensory processing atypicalities are frequent in Autism Spectrum Disorder (ASD) and neurodevelopmental disorders (NDD). Different domains of sensory processing appear to be differentially altered in these disorders. In this study, we explored the sensory profile of two clinical cohorts, in comparison with a sample of typically developing children. METHODS: Behavioral responses to sensory stimuli were assessed using the Sensory Processing Measure (parent-report questionnaire). We included 121 ASD children, 17 carriers of the 16p11.2 deletion (Del 16p11.2) and 45 typically developing (TD) children. All participants were aged between 2 and 12 years. Additional measures included the Tactile Defensiveness and Discrimination Test-Revised, Wechsler Intelligence Scales and Autism Diagnostic Observation Schedule (ADOS-2). Statistical analyses included MANCOVA and regression analyses. RESULTS: ASD children score significantly higher on all SPM subscales compared to TD. Del16p11.2 also scored higher than TD on all subscales except for tactile and olfactory/taste processing, in which they score similarly to TD. When assessing sensory modulation patterns (hyper-, hypo-responsiveness and seeking), ASD did not significantly differ from del16p11.2. Both groups had significantly higher scores across all patterns than the TD group. There was no significant association between the SPM Touch subscale and the TDDT-R. LIMITATIONS: Sensory processing was assessed using a parent-report questionnaire. Even though it captures observable behavior, a questionnaire does not assess sensory processing in all its complexity. The sample size of the genetic cohort and the small subset of ASD children with TDDT-R data render some of our results exploratory. Divergence between SPM Touch and TDDT-R raises important questions about the nature of the process that is assessed. CONCLUSIONS: Touch and olfaction/taste seem to be particularly affected in ASD children compared to del16p11.2. These results indicate that parent report measures can provide a useful perspective on behavioral expression. Sensory phenotyping, when combined with neurobiological and psychophysical methods, might have the potential to provide a better understanding of the sensory processing in ASD and in other NDD.

Skraban-Deardorff syndrome: Six new cases of WDR26-related disease and expansion of the clinical phenotype.

Skraban-Deardorff syndrome (a disease related to variations in the WDR26 gene; OMIM #617616) was first described in a cohort of 15 individuals in 2017. The syndrome comprises intellectual deficiency, severe speech impairment, ataxic gait, seizures, mild hypotonia with feeding difficulties during infancy, and dysmorphic features. Here, we report on six novel heterozygous de novo pathogenic variants in WDR26 in six probands. The patients' phenotypes were consistent with original publication. One patient displayed marked hypotonia with an abnormal muscle biopsy; this finding warrants further investigation. Gait must be closely monitored, in order to highlight any musculoskeletal or neurological abnormalities and prompt further examinations. Speech therapy and alternative communication methods should be initiated early in the clinical follow-up, in order to improve language and oral eating and drinking.

IQSEC2 disorder: A new disease entity or a Rett spectrum continuum?

IQSEC2 mutations are associated with IQSEC2-related intellectual disability (ID). Phenotypic spectrum has been better defined in the last few years by the increasing number of reported cases although the genotype-phenotype relationship for IQSEC2 remains overall complex. As for IQSEC2-related ID a wide phenotypic diversity has been described in Rett syndrome (RTT). Several patients harboring IQSEC2 mutations present with clinical symptoms similar to RTT and some cases meet most of the criteria for classic RTT. With the aim of establishing a genotype-phenotype correlation, we collected data of 16 patients harboring IQSEC2 point mutations (15 of them previously unreported) and of five novel patients carrying CNVs encompassing IQSEC2. Most of our patients surprisingly shared a moderate-to-mild phenotype. The similarities in the clinical course between our mild cases and patients with milder forms of atypical RTT reinforce the hypothesis that also IQSEC2 mutated patients may lay under the wide clinical spectrum of RTT and thus IQSEC2 should be considered in the differential diagnosis. Our data confirm that position, type of variant and gender are crucial for IQSEC2-associated phenotype delineation.