Comprehensive EHMT1 variants analysis broadens genotype-phenotype associations and molecular mechanisms in Kleefstra syndrome.

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.

Expanding the phenotype of Kleefstra syndrome: speech, language and cognition in 103 individuals.

OBJECTIVES: Speech and language impairments are core features of the neurodevelopmental genetic condition Kleefstra syndrome. Communication has not been systematically examined to guide intervention recommendations. We define the speech, language and cognitive phenotypic spectrum in a large cohort of individuals with Kleefstra syndrome. METHOD: 103 individuals with Kleefstra syndrome (40 males, median age 9.5 years, range 1-43 years) with pathogenic variants (52 9q34.3 deletions, 50 intragenic variants, 1 balanced translocation) were included. Speech, language and non-verbal communication were assessed. Cognitive, health and neurodevelopmental data were obtained. RESULTS: The cognitive spectrum ranged from average intelligence (12/79, 15%) to severe intellectual disability (12/79, 15%). Language ability also ranged from average intelligence (10/90, 11%) to severe intellectual disability (53/90, 59%). Speech disorders occurred in 48/49 (98%) verbal individuals and even occurred alongside average language and cognition. Developmental regression occurred in 11/80 (14%) individuals across motor, language and psychosocial domains. Communication aids, such as sign and speech-generating devices, were crucial for 61/103 (59%) individuals including those who were minimally verbal, had a speech disorder or following regression. CONCLUSIONS: The speech, language and cognitive profile of Kleefstra syndrome is broad, ranging from severe impairment to average ability. Genotype and age do not explain the phenotypic variability. Early access to communication aids may improve communication and quality of life.

Growth, body composition, and endocrine-metabolic profiles of individuals with Kleefstra syndrome provide directions for clinical management and translational studies.

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.

Arrhythmias including atrial fibrillation and congenital heart disease in Kleefstra syndrome: a possible epigenetic link.

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.

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient-derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as loss-of-function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.

The impact of lockdown on young people with genetic neurodevelopmental disabilities: a study with the international participatory database GenIDA.

BACKGROUND: Previous publications suggested that lockdown is likely to impact daily living issues of individuals with intellectual disabilities. The authors notably suspected an intensification of behavioural, eating and sleep problems. METHODS: To test these hypotheses, we conducted an international online survey about the impact of COVID-19-associated first lockdown on people with genetic neurodevelopmental disorders. This survey was carried out using GenIDA, an international participatory database collecting medical information on genetic neurodevelopmental disorders. Patients' relatives took part in this online survey from 30/04/2020 to 09/06/2020. This survey adapted from GenIDA standard questionnaire requested information on diagnosis, lifestyle and was based on yes/no answers to questions regarding behaviour, diet, and sleep, in the 6-months period before lockdown and during lockdown. We also asked relatives to evaluate the intensity of these problems by severity level. Finally, relatives could freely comment in open fields on the medical and/or quality of life problems they had encountered during lockdown. RESULTS: In total 199 participants-144 children and 45 adults-with neurodevelopmental disorders (intellectual disability (79.4%) and/or autism spectrum disorder (21.6%)) of various genetic origins, with near-equal male/female (96/103) contribution and originating mainly from Europe and Northern America, were included. The average lockdown duration at time of the survey was 57 days. We did not find differences in the frequency of behavioural, eating and sleep problems before and during lockdown. Moreover, there was no apparent difference in the intensity of eating and sleep disorders between both periods. However, for persons with behavioural problems at both periods, relatives reported an increase in aggressivity, self-aggressivity, depressiveness, stereotypies, and restricted interests during lockdown, all of which might be interpreted as consequences of a lack of stimulation or a reaction to unexpected changes in daily habits. CONCLUSIONS: Our results support previous studies that suggest that the negative impact of lockdown does not depend on the intellectual disability per se but on the associated comorbidities such as behavioural disorders. This study addresses the need for prevention of behavioural disturbance in the vulnerable population with genetic neurodevelopmental disabilities.

Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.

Measuring Adaptive Behavior in Patients with Mendelian Neurodevelopmental Disorders. Comparison of ABAS-3 and Dutch Vineland Scales.

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 0-4/517 ABAS-3 versions would work best to assess the entire spectrum of adaptive functioning adequately.