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Trithorax-related H3K4 methyltransferases, KMT2C and KMT2D, are critical epigenetic modifiers. Haploinsufficiency of KMT2C was only recently recognized as a cause of neurodevelopmental disorder (NDD), so the clinical and molecular spectrums of the KMT2C -related NDD (now designated as Kleefstra syndrome 2) are largely unknown. We ascertained 98 individuals with rare KMT2C variants, including 75 with protein-truncating variants (PTVs). Notably, ∼15% of KMT2C PTVs were inherited. Although the most highly expressed KMT2C transcript consists of only the last four exons, pathogenic PTVs were found in almost all the exons of this large gene. KMT2C variant interpretation can be challenging due to segmental duplications and clonal hematopoesis-induced artifacts. Using samples from 27 affected individuals, divided into discovery and validation cohorts, we generated a moderate strength disorder-specific KMT2C DNA methylation (DNAm) signature and demonstrate its utility in classifying non-truncating variants. Based on 81 individuals with pathogenic/likely pathogenic variants, we demonstrate that the KMT2C -related NDD is characterized by developmental delay, intellectual disability, behavioral and psychiatric problems, hypotonia, seizures, short stature, and other comorbidities. The facial module of PhenoScore, applied to photographs of 34 affected individuals, reveals that the KMT2C -related facial gestalt is significantly different from the general NDD population. Finally, using PhenoScore and DNAm signatures, we demonstrate that the KMT2C -related NDD is clinically and epigenetically distinct from Kleefstra and Kabuki syndromes. Overall, we define the clinical features, molecular spectrum, and DNAm signature of the KMT2C -related NDD and demonstrate they are distinct from Kleefstra and Kabuki syndromes highlighting the need to rename this condition. Pathogenic KMT2C variants cause a disorder, which was named Kleefstra syndrome 2, suggesting its clinical features are highly similar to Kleefstra syndrome. In this study, we delineate the clinical and molecular features of this disorder and show that it is, in fact, different from both Kleefstra and Kabuki syndromes. [ABSTRACT FROM AUTHOR]

The shift to a genotype-first approach in genetic diagnostics has revolutionized our understanding of neurodevelopmental disorders, expanding both their molecular and phenotypic spectra. Kleefstra syndrome (KLEFS1) is caused by EHMT1 haploinsufficiency and exhibits broad clinical manifestations. EHMT1 encodes euchromatic histone methyltransferase-1—a pivotal component of the epigenetic machinery. We have recruited 209 individuals with a rare EHMT1 variant and performed comprehensive molecular in silico and in vitro testing alongside DNA methylation (DNAm) signature analysis for the identified variants. We (re)classified the variants as likely pathogenic/pathogenic (molecularly confirming Kleefstra syndrome) in 191 individuals. We provide an updated and broader clinical and molecular spectrum of Kleefstra syndrome, including individuals with normal intelligence and familial occurrence. Analysis of the EHMT1 variants reveals a broad range of molecular effects and their associated phenotypes, including distinct genotype-phenotype associations. Notably, we showed that disruption of the "reader" function of the ankyrin repeat domain by a protein altering variant (PAV) results in a KLEFS1-specific DNAm signature and milder phenotype, while disruption of only "writer" methyltransferase activity of the SET domain does not result in KLEFS1 DNAm signature or typical KLEFS1 phenotype. Similarly, N-terminal truncating variants result in a mild phenotype without the DNAm signature. We demonstrate how comprehensive variant analysis can provide insights into pathogenesis of the disorder and DNAm signature. In summary, this study presents a comprehensive overview of KLEFS1 and EHMT1, revealing its broader spectrum and deepening our understanding of its molecular mechanisms, thereby informing accurate variant interpretation, counseling, and clinical management. Kleefstra syndrome is a rare neurodevelopmental disorder caused by pathogenic EHMT1 variants affecting multiple organ systems. After almost two decades of research, we describe the current knowledge of EHMT1 variants and their functional consequences, leading to a broad clinical spectrum. Finally, we also obtained important insights into EHMT1 functions. [ABSTRACT FROM AUTHOR]

Objectives Kleefstra Syndrome (KS) is a rare genetic neurodevelopmental disorder caused by a microdeletion in chromosomal region 9q34.3 or a mutation in the euchromatin histone methyltransferase 1 (EHTM1) gene. Patients with KS show a range of clinical symptoms, including delay in motor and speech development, intellectual disability, autistic-like features, childhood hypotonia, and distinctive facial dysmorphic features. The patient is a four-year-old girl who was initially diagnosed with developmental motor delay by a pediatric neurologist and referred to an occupational therapy clinic at the age of six months. The initial assessment showed hypotonia and difficulties with rolling. Occupational therapy intervention was based on principles of neurodevelopmental treatment and sensory integration (SI) with cognitive integration and activities of daily living (ADL) training. With continuous occupational therapy services over more than three years, she overcame many disabilities and improved in occupational performance skills such as gross and fine motor skills as well as cognitive abilities, although her verbal communication skills were not effective. The patient's progress was as follows: she began rolling over at seven months, achieved independent sitting at ten months, crawled at eighteen months, stood with support at twenty months, and took her first steps at twenty-six months. The predominant problem was speech delay, which was noticeable in this syndrome. When a patient is being referred because of KS, occupational and speech therapy assessments should be accurately implemented. [ABSTRACT FROM AUTHOR]

Mendelian neurodevelopmental disorders caused by variants in genes encoding chromatin modification can be categorized as Mendelian disorders of the epigenetic machinery (MDEMs). These disorders have significant overlap in molecular pathways and phenotypes including intellectual disability, short stature, and obesity. Among the MDEMs is Kleefstra syndrome (KLFS), which is caused by haploinsufficiency of EHMT1. Preclinical studies have identified metabolic dysregulation and obesity in KLFS models, but proper clinical translation lacks. In this study, we aim to delineate growth, body composition, and endocrine‐metabolic characteristics in a total of 62 individuals with KLFS. Our results revealed a high prevalence of childhood‐onset overweight/obesity (60%; 28/47) with disproportionately high body fat percentage, which aligns perfectly with previous preclinical studies. Short stature was common (33%), likely due to advanced skeletal maturation. Endocrine‐metabolic investigations showed thyroid dysregulation (22%; 9/41), elevated triglycerides, and decreased blood ammonia levels. Moreover, hand radiographs identified decreased bone mineralization (57%; 8/14) and negative ulnar variance (71%; 10/14). Our findings indicate a high (cardio)metabolic risk in KLFS. Therefore, we recommend monitoring of weight and endocrine‐metabolic profile. Supporting a healthy lifestyle and screening of bone mineralization is advised. Our comprehensive results support translational research and contribute to a better understanding of MDEM‐associated phenotypes. [ABSTRACT FROM AUTHOR]

Objective: Kleefstra syndrome (KS), formerly known as 9q subtelomeric deletion syndrome, is characterized by multiple structural abnormalities. However, most fetuses do not have obvious abnormal phenotypes. In this study, the fetus with KS presented with multiple system structural anomalies, and we aimed to explore the genotype-phenotype correlations of KS fetuses. Case report: Multiple systematic structural anomalies, including severe intrauterine growth restriction (IUGR) and cardiac defects, were detected by ultrasound in the fetus at 33 + 5 weeks' gestation. These abnormalities may be caused by the pathogenic deleted fragment at 9q34.3, including the euchromatic histone methyltransferase 1 (EHMT1) and collagen type V alpha 1 chain (COL5A1) genes, detected by copy number variation sequencing (CNV-seq). Conclusions: It is essential for clinicians to perform CNV-seq combined with multidisciplinary consultation for suspected KS fetuses, especially those with multiple systematic structural anomalies.

Aims Kleefstra syndrome (KS), often diagnosed in early childhood, is a rare genetic disorder due to haploinsufficiency of EHMT1 and is characterized by neuromuscular and intellectual developmental abnormalities. Although congenital heart disease (CHD) is common, the prevalence of arrhythmias and CHD subtypes in KS is unknown. Methods and results Inspired by a novel case series of KS patients with atrial tachyarrhythmias in the USA, we evaluate the two largest known KS registries for arrhythmias and CHD: Radboudumc (50 patients) based on health record review at Radboud University Medical Center in the Netherlands and GenIDA (163 patients) based on worldwide surveys of patient families. Three KS patients (aged 17–25 years) presented with atrial tachyarrhythmias without manifest CHD. In the international KS registries, the median [interquartile range (IQR)] age was considerably younger: GenIDA/Radboudumc at 10/13.5 (12/13) years, respectively. Both registries had a 40% prevalence of cardiovascular abnormalities, the majority being CHD, including septal defects, vascular malformations, and valvular disease. Interestingly, 4 (8%) patients in the Radboudumc registry reported arrhythmias without CHD, including one atrial fibrillation (AF), two with supraventricular tachycardias, and one with non-sustained ventricular tachycardia. The GenIDA registry reported one patient with AF and another with chronic ectopic atrial tachycardia (AT). In total, atrial tachyarrhythmias were noted in six young KS patients (6/213 or 3%) with at least four (three AF and one AT) without structural heart disease. Conclusion In addition to a high prevalence of CHD, evolving data reveal early-onset atrial tachyarrhythmias in young KS patients, including AF, even in the absence of structural heart disease. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome is caused by chromosome 9q34.3 deletion or heterozygous mutations in the euchromatin histone methyl transferase 1 (EHMT1) gene. It can be accompanied by intellectual disability, distinctive facial features, microcephaly, psychiatric disorders, hypotonia in childhood, hearing loss, heart defects, renal defects, epilepsy, speech anomalies, and obesity. Furthermore, genital anomalies are present in 30%–40% of male patients with Kleefstra syndrome, but their mechanisms have not been elucidated. Herein, we report a patient with Kleefstra syndrome presenting with micropenis. The patient was transferred to Kyungpook National University Children's Hospital for management of imperforate anus on the day of birth. Physical examination revealed micropenis with stretched penile length of 0.9 cm and facial dysmorphisms, including hypertelorism and anteverted nares. Chromosomal microarray revealed 424-kb heterozygous deletion at chromosome 9q34.3 (arr[hg19] 9q34.3 (140,234,315-140,659,055)x1). Among the involved main OMIM genes, phenotypically relevant genes were EHMT1 and NSMF. Endocrinological investigation showed low basal gonadotropin and testosterone levels. Anterior pituitary hormones and steroid hormone levels were in the normal range. Testicular function was normal based on human chorionic gonadotropin stimulation test. The patient experienced improvement in penile length growth with intramuscular testosterone enanthate injection initiated at 4 months of age. The purpose of this study is to describe the etiology, endocrine laboratory tests, and treatment of micropenis in Kleefstra syndrome.

Background Kleefstra syndrome (KS) or 9q34.3 microdeletion syndrome (OMIM #610253) is a rare genetic condition featuring intellectual disability, hypotonia, and dysmorphic facial features. Autism spectrum disorder, severe language impairment, and sleep disorders have also been described. The syndrome can be either caused by a microdeletion in 9q34.3 or by pathogenic variants in the euchromatin histone methyltransferase 1 gene (EHMT1 , *607001). Although epilepsy has been reported in 20 to 30% of subjects, a detailed description of epileptic features and underlying etiology is still lacking. The purpose of this study is to investigate epilepsy features in a cohort of epileptic patients with KS. Methods This multicenter study investigated eight patients with KS and epilepsy. Our findings were compared with literature data. Results We included five patients with 9q or 9q34.33 deletions, a subject with a complex translocation involving EHMT1 , and two with pathogenic EHMT1 variants. All patients presented with moderate to severe developmental delay, language impairment, microcephaly, and infantile hypotonia. Although the epileptic manifestations were heterogeneous, most patients experienced focal seizures. The seizure frequency differs according to the age of epilepsy onset, with patients with early-onset epilepsy (before 36 months of age) presenting more frequent seizures. An overtime reduction in seizure frequency, as well as in antiseizure drug number, was observed in all patients. Developmental delay degree did not correlate with seizure onset and frequency or drug resistance. Conclusion Epilepsy is a frequent finding in KS, but the underlying pathogenetic mechanism and specific features remain elusive. [ABSTRACT FROM AUTHOR]

Objective: Several instruments are available for measuring (aspects of) adaptive functioning, but knowledge is lacking about which is best to use to monitor patients with etiologically homogeneous neurodevelopmental disorders. In this study we compare the use of the Vineland-Z and ABAS-3 adaptive behavior scales in such a specific group. Method: Of patients with a molecularly confirmed diagnosis of Kleefstra syndrome, 34 were assessed with both the Vineland-Z and ABAS-3 of which 12 (35,3%) males and 22 (64,7%) females. Raw scores and developmental ages were calculated and a comparison between the instruments was done via correlation analysis. Results: Biological age ranged from 12 to 50 years old (median age of 23,1 ± 9,6 years). Pearson r correlation analyses show that the Vineland-Z and ABAS-3 assessments are highly interchangeable in this population. However, there are practical issues which require attention: (i) the use of ABAS-3 needs several versions to cover the whole adaptive spectrum, and (ii) the Vineland-Z discriminates more at the lower end of the adaptive functioning spectrum compared to the ABAS-3, but less at the higher end. An ideal instrument for this specific purpose is not yet available. Conclusions: We recommend that either the Vineland-Z, with modification of the dated items, the abridged version of the Vineland III, or a merge of the 0-4/5-17 ABAS-3 versions would work best to assess the entire spectrum of adaptive functioning adequately. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome (KS) is a genetic disorder caused by a microdeletion in the 9q34.3 chromosome with related clinical conditions requiring surgical intervention and careful consideration for anesthetic management includ- ing potential difficult airway, cardiac and neurological considerations, pulmonary infections, reflux and delayed gastric emptying, genitourinary abnormalities, hypoto- nia, and seizure disorders. In addition, we observed unique perioperative pain control considerations. We pre- sent a clinical case involving a 2-year-old female who re- ceived anesthesia for robotic ureteral reimplantation sur- gery. Perioperative implications are discussed and con- siderations for anesthetic care provided.

Background: Kleefstra syndrome is a rare genetic condition, which affects at least 1 in 120,000 individuals who have a neurodevelopmental disorder, characterized by the core clinical phenotype of intellectual disability, hypotonia, severe speech delay, and distinct facial characteristics with additional clinical features including sleep disturbance, overweight, psychiatric disorders, and autism spectrumdisorder. To date, a limited number of case reports of Kleefstra syndrome with psychiatric manifestations have been reported. Case presentation: We reported a case of a 35-year-old male diagnosed with Kleefstra syndrome, who also had diagnoses of autism spectrum disorder and moderate to severe intellectual disability. He exhibited various psychiatric manifestations, including temporarily manic-like symptoms, excessive eating/overweight, addictive/gambling behaviors, inappropriate and unsafe internet use, sleep disturbance, rigid routines, and behaviors that challenged in the form of meltdowns. These symptoms were eventually relatively successfully managed with a combination of non-pharmacological and pharmacological treatments. Conclusion: To our knowledge, there is only a limited number of case reports that detail patients with Kleefstra syndrome exhibiting various psychiatric manifestations. Our report adds further knowledge to the paucity of literature and highlights the effectiveness of a combination of non-pharmacological and pharmacological treatments for behavioral/psychiatric dificulties in Kleefstra syndrome. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome (KS) is a genetic disorder caused by a microdeletion in the 9q34.3 chromosome with related clinical conditions requiring surgical intervention and careful consideration for anesthetic management including potential difficult airway, cardiac and neurological considerations, pulmonary infections, reflux and delayed gastric emptying, genitourinary abnormalities, hypotonia, and seizure disorders. In addition, we observed unique perioperative pain control considerations. We present a clinical case involving a 2-year-old female who received anesthesia for robotic ureteral reimplantation surgery. Perioperative implications are discussed and considerations for anesthetic care provided. [ABSTRACT FROM AUTHOR]

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction.

BackgroundKleefstra syndrome is a rare genetic condition, which affects at least 1 in 120,000 individuals who have a neurodevelopmental disorder, characterized by the core clinical phenotype of intellectual disability, hypotonia, severe speech delay, and distinct facial characteristics with additional clinical features including sleep disturbance, overweight, psychiatric disorders, and autism spectrum disorder. To date, a limited number of case reports of Kleefstra syndrome with psychiatric manifestations have been reported.Case presentationWe reported a case of a 35-year-old male diagnosed with Kleefstra syndrome, who also had diagnoses of autism spectrum disorder and moderate to severe intellectual disability. He exhibited various psychiatric manifestations, including temporarily manic-like symptoms, excessive eating/overweight, addictive/gambling behaviors, inappropriate and unsafe internet use, sleep disturbance, rigid routines, and behaviors that challenged in the form of meltdowns. These symptoms were eventually relatively successfully managed with a combination of non-pharmacological and pharmacological treatments.ConclusionTo our knowledge, there is only a limited number of case reports that detail patients with Kleefstra syndrome exhibiting various psychiatric manifestations. Our report adds further knowledge to the paucity of literature and highlights the effectiveness of a combination of non-pharmacological and pharmacological treatments for behavioral/psychiatric difficulties in Kleefstra syndrome.

Introduction. Autism Spectrum Disorders (ASD) are a group of neurodevelopmental disorders that manifest in early childhood but persist into adolescence and adulthood. Kleefstra syndrome is found in 500 cases worldwide and is characterised by distinct facial features, developmental delay, mental retardation, hypotonia, communication difficulties, behavioural and socialisation disorders. The aim of the study is to analyse the genetic aspects, clinical polymorphism and developmental features of Kleefstra syndrome in children through the lens of a clinical case. Material and methods: A clinical case with clinical symptoms suggestive of a genetic pathology was evaluated at IMSP Institute of Mother and Child in April 2023. Examinations included, brain MRI imaging, molecular-genetic examination by genomic sequencing. Genetic testing was carried out by whole genome sequencing (NGS), Next Generation Sequencing (NGS) technique. Results. One clinical case was reported: a boy, aged 8 years and 2 months, who was referred for cognitive disorders, mainly in the area of speech and language and behavioural disorders. Examination findings: multiple disembryogenic stigmata, microcephaly, clinical signs of ASD (defective social interaction, stereotyped movements, communication disorders, repetitive rocking movements), behavioural disorders (hyperagitation), cognitive-verbal retardation (learning disorders, defective speech, IQ test = 38 points), also multiple disembryogenic stigmata, motor clumsiness. Genetic testing: submicroscopic 9q34.3 deletion, mutations in EHMT1 gene (suggestive of Kleefstra syndrome). Conclusions. ASD may be associated with genetic diseases. One such disorder is Kleefstra syndrome, a rare genetic disorder characterized by craniofacial dysmorphism, developmental delay, hypotonia, mental retardation, behavioural and socialization disorders. Thus, ASD needs to be screened to exclude associated genetic diseases. [ABSTRACT FROM AUTHOR]

Novel approaches to uncover the molecular etiology of neurodevelopmental disorders (NDD) are highly needed. Even using a powerful tool such as whole exome sequencing (WES), the diagnostic process may still prove long and arduous due to the high clinical and genetic heterogeneity of these conditions. The main strategies to improve the diagnostic rate are based on family segregation, re-evaluation of the clinical features by reverse-phenotyping, re-analysis of unsolved NGS-based cases and epigenetic functional studies. In this article, we described three selected cases from a cohort of patients with NDD in which trio WES was applied, in order to underline the typical challenges encountered during the diagnostic process: (1) an ultra-rare condition caused by a missense variant in MEIS2, identified through the updated Solve-RD re-analysis; (2) a patient with Noonan-like features in which the NGS analysis revealed a novel variant in NIPBL causing Cornelia de Lange syndrome; and (3) a case with de novo variants in genes involved in the chromatin-remodeling complex, for which the study of the epigenetic signature excluded a pathogenic role. In this perspective, we aimed to (i) provide an example of the relevance of the genetic re-analysis of all unsolved cases through network projects on rare diseases; (ii) point out the role and the uncertainties of the reverse phenotyping in the interpretation of the genetic results; and (iii) describe the use of methylation signatures in neurodevelopmental syndromes for the validation of the variants of uncertain significance. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome is a rare genetic disease characterized by mental retardation, hypotonia, and a characteristic facial appearance. Furthermore, in some cases, Kleefstra syndrome is associated with various anorectal and genitourinary complications, including imperforated anus, vesicoureteral reflux, hydronephrosis, and chronic kidney disease. Herein, we present a case of Kleefstra syndrome with recurrent urinary tract infections associated with vesicoureteral reflux and rectourethral fistula, which was treated by a multidisciplinary approach.

Challenging behaviors maintained by sensory consequences (i.e., automatic reinforcement) can be difficult to treat without constant supervision. This type of time commitment can be extremely difficult for families to do effectively. Competing stimulus-based treatments have shown promise for treating challenging behavior during periods of low supervision as they require minimal interaction with the child. Accordingly, families may benefit from learning to become self-sufficient in using this approach to assessment and treatment. This study evaluated the effects of a parent-conducted assessment and treatment on the challenging behavior of destructive paper tearing by a girl with Kleefstra Syndrome (9q34.3 deletion syndrome). Following a parent-conducted functional analysis, the mother completed an abbreviated competing stimulus assessment (CSA), and then delivered a treatment involving concurrent availability of all items identified in her daughter's CSA. Results showed that the treatment was associated with generalized, reduction in challenging behavior that maintained in follow-up. [ABSTRACT FROM AUTHOR]

Abstract Background Over 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays. Methods In this study we test a rapid genetic variant assessment pipeline using CRISPR homology directed repair to introduce single nucleotide variants into inducible pluripotent stem cells (iPSCs), followed by neuronal disease modelling, and functional genomics on amplicon and RNA sequencing, to determine cellular changes to support patient diagnosis and identify disease mechanism. Results As proof-of-principle, we investigated an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 c.3430C > T; p.Gln1144*) genetic variant pathogenic for Kleefstra syndrome and determined changes in gene expression during neuronal progenitor cell differentiation. This pipeline rapidly identified Kleefstra syndrome in genetic variant cells compared to healthy cells, and revealed novel findings potentially implicating the key transcription factors REST and SP1 in disease pathogenesis. Conclusion The study pipeline is a rapid, robust method for genetic variant assessment that will support rare diseases patient diagnosis. The results also provide valuable information on genome wide perturbations key to disease mechanism that can be targeted for drug treatments.

Objective: to report the first case series of Brazilian children diagnosed with Kleefstra syndrome, present a possible phenotype expansion to the syndrome and to raise physicians' awareness for this rare disease., Results: seven patients with confirmed KS were evaluated, including 5 males and 2 females. Abnormal prenatal findings were observed in 4 patients. Most patients were born at term, with normal birth measurements. All patients had neurodevelopmental delay and 6 evolved with intellectual disability. Hearing loss was present in 57.1% of patients and 28.7% had congenital heart disease. In males, cryptorchidism was present in 75%. Despite the facial dysmorphisms, only 2 out of 7 patients had a pre-test clinical suspicion of KS. One specific patient presented bilateral agenesis of the semicircular canals, a very rare ear manifestation in Kleefstra syndrome, representing a possible phenotype expansion of the syndrome., Conclusion: this report aims to promote awareness among physicians evaluating patients in a context of neurodevelopmental delay or congenital malformations, especially congenital heart defects. We also highlight a possible phenotype expansion of the syndrome, with a case of semicircular anomaly, not reported in this syndrome so far., Competing Interests: Declaration of competing interest nothing to declare., (Copyright © 2024. Published by Elsevier Masson SAS.)

Novel approaches to uncover the molecular etiology of neurodevelopmental disorders (NDD) are highly needed. Even using a powerful tool such as whole exome sequencing (WES), the diagnostic process may still prove long and arduous due to the high clinical and genetic heterogeneity of these conditions. The main strategies to improve the diagnostic rate are based on family segregation, re-evaluation of the clinical features by reverse-phenotyping, re-analysis of unsolved NGS-based cases and epigenetic functional studies. In this article, we described three selected cases from a cohort of patients with NDD in which trio WES was applied, in order to underline the typical challenges encountered during the diagnostic process: (1) an ultra-rare condition caused by a missense variant in MEIS2, identified through the updated Solve-RD re-analysis; (2) a patient with Noonan-like features in which the NGS analysis revealed a novel variant in NIPBL causing Cornelia de Lange syndrome; and (3) a case with de novo variants in genes involved in the chromatin-remodeling complex, for which the study of the epigenetic signature excluded a pathogenic role. In this perspective, we aimed to (i) provide an example of the relevance of the genetic re-analysis of all unsolved cases through network projects on rare diseases; (ii) point out the role and the uncertainties of the reverse phenotyping in the interpretation of the genetic results; and (iii) describe the use of methylation signatures in neurodevelopmental syndromes for the validation of the variants of uncertain significance.

Background: Over 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays. Methods: In this study we test a rapid genetic variant assessment pipeline using CRISPR homology directed repair to introduce single nucleotide variants into inducible pluripotent stem cells (iPSCs), followed by neuronal disease modelling, and functional genomics on amplicon and RNA sequencing, to determine cellular changes to support patient diagnosis and identify disease mechanism. Results: As proof-of-principle, we investigated an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 c.3430C > T; p.Gln1144*) genetic variant pathogenic for Kleefstra syndrome and determined changes in gene expression during neuronal progenitor cell differentiation. This pipeline rapidly identified Kleefstra syndrome in genetic variant cells compared to healthy cells, and revealed novel findings potentially implicating the key transcription factors REST and SP1 in disease pathogenesis. Conclusion: The study pipeline is a rapid, robust method for genetic variant assessment that will support rare diseases patient diagnosis. The results also provide valuable information on genome wide perturbations key to disease mechanism that can be targeted for drug treatments. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome (KS) is a rare autosomic dominant genetic disorder caused by euchromatic histone methyltransferase 1 (EHMT1) alterations. Patients mainly present with moderate to severe intellectual disability, a severe delay in/or absence of speech, autism spectrum disorder, childhood hypotonia, neuropsychiatric anomalies, and distinctive dysmorphic features. Here, we report the cases of a male and a female, two younger siblings of three, with asymptomatic parents. An EHMT1 new mutation was identified. Both presented with a typical core phenotype. Some specific features were noted, such as macrocephaly (previously reported) and enuresis (not yet described). Parental analysis identified the mutation in the mosaic state in the father. Reverse phenotyping enabled us to highlight the pauci phenotype features of inguinal hernia, azoospermia, and possible behavioral disorders. This allowed us to adapt his follow‐up and genetic counseling for the family. Our three reported cases provide a new description of KS with an intragenic EHMT1 mutation, whereas in the literature most reported cases have EHMT1 deletions. Moreover, in the areas of next‐generation sequencing and trio techniques with parental segregation, it is important to remain cautious about disregarding variants based on an autosomal recessive hypothesis. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome is a genetic disorder that may involve different parts of the body, but its main characteristics are intellectual disability and childhood hypotonia. We report a 10-year-old mentally retarded male patient who presented with seizure. His medical history revealed recurrent upper respiratory infections, neurodevelopmental delay, and epilepsy. It was also found that he had had a hospitalization in the neonatal intensive care unit for five days due to tachypnea, low APGAR score, and meconium aspiration syndrome. His brain MRI had shown some degree of distension of the lateral cerebral ventricles with the basal cistern. The electromyography and the nerve conduction velocity were however normal. He was diagnosed with Kleefstra syndrome by the loss of the EHMT1 gene. He is now under treatment by piracetam and work-therapy. This is the second case report of this syndrome in Iran. This case presentation aims to improve the diagnosis of Kleefstra syndrome patients, as a rare syndrome with non-characteristic manifestations.

Kleefstra syndrome (KS) is a genetic syndrome caused by a haploinsufficiency of the EHMT1 gene and characterized by intellectual disability, language disorders, childhood hypotonia and distinct facial features. Only a few cases of first episode of psychosis in KS have already been reported. We describe a young female patient with KS who presented a first episode of psychosis. In a context of an initial diagnosis wavering and a lack of recommendations, this clinical observation illustrates the importance of psychiatric comorbidities and their diagnostic and therapeutic complexity in KS; with a need for multidisciplinary management considering its specific aspects and vulnerabilities. [ABSTRACT FROM AUTHOR]

Case: A boy with bilateral congenital clubfoot, Kleefstra syndrome, and Duchenne muscular dystrophy (DMD) developed clubfoot relapse after excellent initial correction with the Ponseti method and maintenance abduction bracing. A traditional clubfoot tibialis anterior transfer was augmented with a tibialis posterior tendon transfer, given underlying DMD at ages 7 and 10 years for the right foot and left foot, respectively. Conclusion: This case illustrates successful maintenance of correction using combined tibialis anterior and tibialis posterior tendon transfer. Tibialis posterior tendon transfer may be useful for clubfoot relapse in conditions that weaken the tibialis anterior or in failed tibialis anterior tendon transfers. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome (KS) is a rare genetic disorder characterized by developmental delay/intellectual disability, generalized hypotonia, neuropsychiatric anomalies, and a distinctive facial appearance. These syndromal manifestations were recently found to be caused by a 9q34.3 gene microdeletion or mutation in the EHMT1 gene, a gene responsible for chromatin remodeling during neurodevelopment and homeostatic plasticity. KS was first described in 2005; since then, diagnostic advancements have narrowed diagnostic criterion and improved identification of this syndrome. In this case report, we discuss a 30 year old male diagnosed following rapid neurocognitive deterioration who was diagnosed with KS after a 16kb deletion of chromosome 9q34.3 was found on chromosomal microarray. In review of his case material it was clear that characteristic signs occurred in adolescence. The current literature regarding KS from a psychiatric approach is reviewed to define specific behavioral manifestations and to delineate current recommended treatments with a focus on earlier identification and intervention. Through expanding microarray technologies KS is expected to become more frequently diagnosed; attention to its manifestation in adolescence may provide optimal adolescent and emerging adulthood care at the intersection of psychiatry and general medicine. [ABSTRACT FROM AUTHOR]

Mutations in the Euchromatic Histone Methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a rare form of intellectual disability (ID) with strong autistic traits and sensory processing deficits. Proper development of inhibitory interneurons is crucial for sensory function. Here we report a timeline of Parvalbumin-positive (PV+) interneuron development in the three most important sensory cortical areas in the Ehmt1+/− mouse. We find a hitherto unreported delay of PV+ neuron maturation early in sensory development, with layer- and region-specific variability later in development. The delayed PV+ maturation is also reflected in a delayed maturation of GABAergic transmission in Ehmt1+/− auditory cortex, where we find a reduced GABA release probability specifically in putative PV+ synapses. Together with earlier reports of excitatory impairments in Ehmt1+/− neurons, we propose a shift in excitatory-inhibitory balance towards overexcitability in Ehmt1+/− sensory cortices as a consequence of early deficits in inhibitory maturation. [ABSTRACT FROM AUTHOR]

Abstract Background Genetic mosaicism is only detected occasionally when there are no obvious health or developmental issues. Most cases concern healthy parents in whom mosaicism is identified upon targeted testing of a genetic defect that was initially detected in their children. A germline genetic defect affecting the euchromatin histone methyltransferase 1 (EHMT1) gene causes Kleefstra syndrome, which is associated with the typical triad of distinct facial appearance, (childhood) hypotonia, and intellectual disability. A high degree of psychopathology is associated with this syndrome. A few parents with a mosaic EHMT1 mutation have been detected upon testing after a child was diagnosed with a germline EHMT1 defect. At first glance, carriers of a mosaic EHMT1 mutation appeared to function normally. However, recent studies have shown that de novo, postzygotic mutations in important developmental genes significantly contribute to autism spectrum disorder (ASD). Therefore, we hypothesized that EHMT1 mosaicism could cause neuropsychiatric defects. To investigate this, we performed a detailed investigation of cognitive neuropsychiatric parameters in parents identified with EHMT1 mosaicism. Methods Three adults (two males, one female) with a genetically confirmed diagnosis of EHMT1 mosaicism were examined by means of a battery of tests and observational instruments covering both neurocognitive and psychiatric features. The battery included the following instruments: the Autism Diagnostic Observation Schedule (ADOS), the mini Psychiatric Assessment Schedules for Adults with Developmental Disabilities (mini PAS-ADD), the Vineland Adaptive Behavior Scales (VABS), and the Cambridge Neuropsychological Test Automated Battery (CANTAB). These measures were compared with our previously reported data from Kleefstra syndrome patients with confirmed (germline) EHMT1 defects. Results All three subjects achieved maximum total scores on the VABS, indicative of adequate (adaptive) functioning. In all, scores above cutoff were found on the ADOS for ASD and on the mini PAS-ADD for major depressive disorder (lifetime). Finally, results on the CANTAB showed impaired cognitive flexibility in all subjects. Conclusion Individuals with EHMT1 mosaicism seem to have increased vulnerability for developing severe psychopathology, especially ASD and mood disorders. Although at first glance they appear to be well-adapted in their daily functioning, they may experience significant psychiatric symptoms and show reduced cognitive flexibility in comparison to the general population.

Spine deformities could be considered a possible manifestation of the childhood hypotonia, typical feature of Kleefstra Syndrome (KS). There is a paucity of literature describing posterior spinal fusion in the Kleefstra syndrome patient. For patients who develop severe scoliotic curve, bracing is often ineffective and surgery is recommended. We report the first corrective surgery for scoliosis in one patient with KS. We describe a case of 13-year-old female with severe developmental scoliosis in KS. Preoperative examination showed a thoracolumbar scoliosis with left convex thoracic curve (T3-T9, 97°) and right convex thoracolumbar curve (T9-L3, 88°). Posterior correction, pedicle screw fixation and bone graft fusion T3-L5 was performed. Postoperatively, the thoracic curve was corrected to 33° while the thoracolumbar one to 26° and better standing posture was obtained. Six month follow-up images showed no loosening of the hardware. The patient is still in our follow-up program. Scoliosis seems to be a rare evenience of the severe hypotonia of patients with KS. We report the first case of scoliosis in KS treated successfully with surgery. Corrective surgery for spinal deformity, such as scoliosis, could help in posture and improve the quality of life especially in complicated patients such as syndromic ones. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome is a rare neurogenetic disorder caused by a subtelomeric 9q34.3 deletion or by an intragenic mutation of the euchromatin histone methyl transferase 1 gene (EHMT1). Approximately 20% to 30% of individuals have hearing loss. The left temporal bone of one subject with hearing loss was studied using light microscopy. There were several abnormalities including dysostosis of the stapes without fixation, enlarged vestibular aqueduct, anomalies of the organ of Corti in the basal turn, cyst formation in the stria vascularis, and dysmorphia of the cochlear modiolus and the vestibular labyrinth. This is the first published description of the otopathology in Kleefstra syndrome. Laryngoscope, 130:2028-2033, 2020. [ABSTRACT FROM AUTHOR]

Kleefstra syndrome is a genetic disorder that may involve different parts of the body, but its main characteristics are intellectual disability and childhood hypotonia. We report a 10-yearold mentally retarded male patient who presented with seizure. His medical history revealed recurrent upper respiratory infections, neurodevelopmental delay, and epilepsy. It was also found that he had had a hospitalization in the neonatal intensive care unit for five days due to tachypnea, low APGAR score, and meconium aspiration syndrome. His brain MRI had shown some degree of distension of the lateral cerebral ventricles with the basal cistern. The electromyography and the nerve conduction velocity were however normal. He was diagnosed with Kleefstra syndrome by the loss of the EHMT1 gene. He is now under treatment by piracetam and work-therapy. This is the second case report of this syndrome in Iran. This case presentation aims to improve the diagnosis of Kleefstra syndrome patients, as a rare syndrome with non-characteristic manifestations. [ABSTRACT FROM AUTHOR]

The type 2 lysine methyltransferases KMT2C and KMT2D are large, enzymatically active scaffold proteins that form the core of nuclear regulatory structures known as KMT2C/D COMPASS complexes (complex of proteins associating with Set1). These evolutionarily conserved proteins regulate DNA promoter and enhancer elements, modulating the activity of diverse cell types critical for embryonic morphogenesis, central nervous system development, and post-natal survival. KMT2C/D COMPASS complexes and their binding partners enhance active gene expression of specific loci via the targeted modification of histone-3 tail residues, in general promoting active euchromatic conformations. Over the last 20 years, mutations in five key COMPASS complex genes have been linked to three human congenital syndromes: Kabuki syndrome (type 1 [KMT2D] and 2 [KDM6A]), Rubinstein-Taybi syndrome (type 1 [CBP] and 2 [EP300]), and Kleefstra syndrome type 2 (KMT2C). Here, we review the composition and biochemical function of the KMT2 complexes. The specific cellular and embryonic roles of the KMT2C/D COMPASS complex are highlight with a focus on clinically relevant mechanisms sensitive to haploinsufficiency. The phenotypic similarities and differences between the members of this new family of disorders are outlined and emerging therapeutic strategies are detailed. [ABSTRACT FROM AUTHOR]

Aims: Kleefstra syndrome (KS), often diagnosed in early childhood, is a rare genetic disorder due to haploinsufficiency of EHMT1 and is characterized by neuromuscular and intellectual developmental abnormalities. Although congenital heart disease (CHD) is common, the prevalence of arrhythmias and CHD subtypes in KS is unknown., Methods and Results: Inspired by a novel case series of KS patients with atrial tachyarrhythmias in the USA, we evaluate the two largest known KS registries for arrhythmias and CHD: Radboudumc (50 patients) based on health record review at Radboud University Medical Center in the Netherlands and GenIDA (163 patients) based on worldwide surveys of patient families. Three KS patients (aged 17-25 years) presented with atrial tachyarrhythmias without manifest CHD. In the international KS registries, the median [interquartile range (IQR)] age was considerably younger: GenIDA/Radboudumc at 10/13.5 (12/13) years, respectively. Both registries had a 40% prevalence of cardiovascular abnormalities, the majority being CHD, including septal defects, vascular malformations, and valvular disease. Interestingly, 4 (8%) patients in the Radboudumc registry reported arrhythmias without CHD, including one atrial fibrillation (AF), two with supraventricular tachycardias, and one with non-sustained ventricular tachycardia. The GenIDA registry reported one patient with AF and another with chronic ectopic atrial tachycardia (AT). In total, atrial tachyarrhythmias were noted in six young KS patients (6/213 or 3%) with at least four (three AF and one AT) without structural heart disease., Conclusion: In addition to a high prevalence of CHD, evolving data reveal early-onset atrial tachyarrhythmias in young KS patients, including AF, even in the absence of structural heart disease., Competing Interests: Conflict of interest: S.M.N. reports consulting support from Abbott Inc., UpToDate Inc., LifeSignals Inc., and TDK Inc., intellectual property owned by University of California Regents and Stanford University. P.J.W. is a consultant for EpiEndoAF. N.B. reports speaker fees from Abbott and Zoll. M.V.P. has grant funding support from Apple Inc., consulting support from Biotronik and Boston Scientific, and consulting and equity interest in QALY, Inc. All remaining authors have declared no conflicts of interest or disclosures., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

Kleefstra Syndrome (KS) is a rare monogenetic syndrome, caused by haploinsufficiency of the euchromatic histone methyl transferase 1 (EHMT1) gene, an important regulator of neurodevelopment. The clinical features of KS include intellectual disability, autistic behavior and gastrointestinal problems. The gut microbiota, an important modifier of the gut-brain-axis, may constitute an unexplored mechanism underlying clinical KS variation. We investigated the gut microbiota composition of 23 individuals with KS (patients) and 40 of their family members, to test whether (1) variation in the gut microbiota associates with KS diagnosis and (2) variation within the gut microbiota relates with KS syndrome symptoms. Both alpha and beta diversity of patients were different from their family members. Genus Coprococcus 3 was lower in abundance in patients compared to family members. Moreover, abundance of genus Merdibacter was lower in patients versus family members, but only in participants reporting intestinal complaints. Within the patient group, behavioral problems explained 7% of beta diversity variance. Also, within this group, we detected higher levels of Atopobiaceae - uncultured and Ruminococcaceae Subdoligranulum associated with higher symptom severity. These significant signatures in the gut microbiota composition in patients with KS suggest that microbiota differences are part of the KS phenotype., (© 2023 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics published by Wiley Periodicals LLC.)

Both copy number losses and gains occur within subtelomeric 9q34 region without common breakpoints. The microdeletions cause Kleefstra syndrome (KS), whose responsible gene is EHMT1. A 9q34 duplication syndrome (9q34 DS) had been reported in literature, but it has never been characterized by a detailed molecular analysis of the gene content and endpoints. To the best of our knowledge, we report on the first patient carrying the smallest 9q34.3 duplication containing EHMT1 as the only relevant gene. We compared him with 21 reported patients described here as carrying 9q34.3 duplications encompassing the entire gene and extending within ~ 3 Mb. By surveying the available clinical and molecular cytogenetic data, we were able to discover that similar neurodevelopmental disorders (NDDs) were shared by patient carriers of even very differently sized duplications. Moreover, some facial features of the 9q34 DS were more represented than those of KS. However, an accurate in silico analysis of the genes mapped in all the duplications allowed us to support EHMT1 as being sufficient to cause a NDD phenotype. Wider patient cohorts are needed to ascertain whether the rearrangements have full causative role or simply confer the susceptibility to NDDs and possibly to identify the cognitive and behavioral profile associated with the increased dosage of EHMT1. [ABSTRACT FROM AUTHOR]

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Kleefstra syndrome (KS) is a rare genetic condition resulting from either 9q34.3 microdeletions or mutations in the EHMT1 gene located in the same genomic region. To date, approximately 100 patients have been reported, thereby allowing the core phenotype of KS to be defined as developmental delay/intellectual disability, generalized hypotonia, neuropsychiatric anomalies, and a distinctive facial appearance. Here, to further expand the knowledge on genotype and phenotype of this condition, we report 2 novel cases: one patient carrying a 46-kb 9q34.3 deletion and showing macrocephaly never described in KS, and a second patient carrying a classic 9q34.3 deletion, presenting with a previously unreported skeletal feature (postaxial polydactyly of the right foot) and an unusual brain anomaly (olfactory bulb hypoplasia) observed via magnetic resonance imaging. Further, we provide a review of the current literature regarding KS and compare these 2 patients with those previously described, thereby confirming that the genotype-phenotype correlation in KS remains difficult to determine. [ABSTRACT FROM AUTHOR]

EHMT1 is an epigenetic factor with histone methyltransferase activity that appears mutated in Kleefstra syndrome, a neurodevelopmental genetic disorder characterized by developmental delay, intellectual disability, and autistic-like features. Despite recent progress in the study of the function of this gene and the molecular etiology of the disease, our knowledge of how EHMT1 haploinsufficiency causes Kleefstra syndrome is still very limited. Here, we show that EHMT1 depletion in RPE1 cells leads to alterations in the morphology and distribution of different subcellular structures, such as the Golgi apparatus, the lysosomes and different cell adhesion components. EHMT1 downregulation also increases centriolar satellites detection, which may indicate a role for EHMT1 in centrosome functioning. Furthermore, the migration process is also altered in EHMT1 depleted cells, which show reduced migration capacity. We consider that the described phenotypes could open new possibilities for understanding the functional impact of EHMT1 haploinsufficiency in Kleefstra syndrome, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in this neurodevelopmental disorder. This knowledge could be relevant not only for the treatment of this syndrome, but also for other neurodevelopmental conditions that could share similar deregulated cellular pathways. • EHMT1 is necessary for the maintenance of the Golgi apparatus structure. • Downregulation of EHMT1 leads to lysosomes accumulation. • Reduced levels of EHMT1 induce changes in cell adhesion. • The increased detection of centriolar satellites after EHMT1 depletion suggests a role for EHMT1 in centrosome functioning. • EHMT1 depleted cells show reduced migration capacity. [ABSTRACT FROM AUTHOR]

Kleefstra Syndrome is characterized by severe mental retardation, brachycephaly, microcephaly, epileptic seizures, distinct facial features, and infantile weak muscle tone and heart defects. Deletion of EHMT1 is the main player in 75% of cases. Because of blurriness in genotype-phenotype correlation through clinical and molecular features of both 9q34.3 microdeletion patients and those with an intragenic EHMT1 mutation in Kleefstra Syndrome, genetic characterization of patients with clinical symptoms of such spectrum is desirable. We report the first Kleefstra Syndrome patient in Iran characterized through genetic approaches. Our report could improve KS diagnosis in Iran and prepare PND and PGs options for involved families.