BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation.
RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain.
CONCLUSIONS: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Helsmoortel-Van der Aa syndrome (HVDAS) is a rare genetic disorder caused by variants in the activity-dependent neuroprotector homeobox (ADNP) gene; hence, it is also called ADNP syndrome. ADNP is a multitasking protein with the function as a transcription factor, playing a critical role in brain development. Furthermore, ADNP variants have been identified as one of the most common single-gene causes of autism spectrum disorder (ASD) and intellectual disability.
We assembled a cohort of 15 Chinese pediatric patients, identified 13 variants in the coding region of ADNP gene, and evaluated their clinical phenotypes. Additionally, we constructed the corresponding ADNP variants and performed western blotting and immunofluorescence analysis to examine their protein expression and subcellular localization in human HEK293T and SH-SY5Y cells.
Our study conducted a thorough characterization of the clinical manifestations in 15 children with ADNP variants, and revealed a broad spectrum of symptoms including global developmental delay, intellectual disability, ASD, facial abnormalities, and other features. In vitro studies were carried out to check the expression of ADNP with identified variants. Two cases presented missense variants, while the remainder exhibited nonsense or frameshift variants, leading to truncated mutants in in vitro overexpression systems. Both overexpressed wildtype ADNP and all the different mutants were found to be confined to the nuclei in HEK293T cells; however, the distinctive pattern of nuclear bodies formed by the wildtype ADNP was either partially or entirely disrupted by the mutant proteins. Moreover, two variants of p.Y719* on the nuclear localization signal (NLS) of ADNP disrupted the nuclear expression pattern, predominantly manifesting in the cytoplasm in SH-SY5Y cells.
Our study was limited by a relatively small sample size and the absence of a longitudinal framework to monitor the progression of patient conditions over time. Additionally, we lacked in vivo evidence to further indicate the causal implications of the identified ADNP variants.
Our study reported the first cohort of HVDAS patients in the Chinese population and provided systematic clinical presentations and laboratory examinations. Furthermore, we identified multiple genetic variants and validated them in vitro. Our findings offered valuable insights into the diverse genetic variants associated with HVDAS.

UNASSIGNED: Helsmoortel-van der Aa syndrome, also known as ADNP syndrome, is a condition that causes developmental delay, language impairment, autism spectrum, and variable extraneurologic features. It is caused by heterozygous mutations in the ADNP gene on chromosome 20q13. Most of the genetic causes of Helsmoortel-van der Aa syndrome have been reported are as de novo nonsense or frameshift stop mutations in exon 5 of ADNP gene, while fewer truncating variants were discovered in exons 4 and the 5\' end of exon 5.
UNASSIGNED: In our study, a 4-year-old female Chinese patient was reported with delayed psychomotor development, language impairment, ataxia, anxiety, aggressive behavior, and congenital heart defect. Trio whole exome sequencing and copy number variation sequencing were performed.
UNASSIGNED: A novel de novo heterozygous pathogenic mutation c.568C > T (p.Gln190Ter) was identified in the ADNP gene of the proband. His unaffected parents did not have the variant. According to the American College of Medical Genetics (ACMG) guidelines, c.568C > T was classified as \"pathogenic\".
UNASSIGNED: Our report indicated that c.568C > T (p.Gln190Ter) in ADNP gene is the cause of abnormal development of the nervous system, congenital heart disease and strabismus, broadening the spectrum of ADNP gene mutations associated with Helsmoortel-van der Aa syndrome.

Individuals affected with autism often suffer additional co-morbidities such as intellectual disability. The genes contributing to autism cluster on a relatively limited number of cellular pathways, including chromatin remodeling. However, limited information is available on how mutations in single genes can result in such pleiotropic clinical features in affected individuals. In this review, we summarize available information on one of the most frequently mutated genes in syndromic autism the Activity-Dependent Neuroprotective Protein (ADNP).
Heterozygous and predicted loss-of-function ADNP mutations in individuals inevitably result in the clinical presentation with the Helsmoortel-Van der Aa syndrome, a frequent form of syndromic autism. ADNP, a zinc finger DNA-binding protein has a role in chromatin remodeling: The protein is associated with the pericentromeric protein HP1, the SWI/SNF core complex protein BRG1, and other members of this chromatin remodeling complex and, in murine stem cells, with the chromodomain helicase CHD4 in a ChAHP complex. ADNP has recently been shown to possess R-loop processing activity. In addition, many additional functions, for instance, in association with cytoskeletal proteins have been linked to ADNP.
We here present an integrated evaluation of all current aspects of gene function and evaluate how abnormalities in chromatin remodeling might relate to the pleiotropic clinical presentation in individual\"s\" with Helsmoortel-Van der Aa syndrome.

The ADNP-gene-related neurodevelopmental disorder Helsmoortel-Van der Aa syndrome is a rare syndromic-intellectual disability-an autism spectrum disorder first described by Helsmoortel and Van der Aa in 2014. Recently, a large cohort including 78 patients and their detailed phenotypes were presented by Van Dijck et al., 2019, who reported developmental delay, speech delay and autism spectrum disorder as nearly constant findings with or without variable cardiological, gastroenterological, urogenital, endocrine and neurological manifestations. Among cardiac malformations, atrial septal defect, patent ductus arteriosus, patent foramen ovale and mitral valve prolapse were the most common findings, but other unspecified defects, such as mild pulmonary valve stenosis, were also described. We present two patients with pathogenic ADNP variants and unusual cardiothoracic manifestations-Bland-White-Garland syndrome, pectus carinatum superiorly along the costochondral junctions and pectus excavatum inferiorly in one patient, and Kawasaki syndrome with pericardiac effusion, coronary artery dilatation and aneurysm in the other-who were successfully treated with intravenous immunoglobulin, corticosteroid and aspirin. Both patients had ectodermal and/or skeletal features overlapping those seen in RASopathies, supporting the observations of Alkhunaizi et al. 2018. on the clinical overlap between Helsmoortel-Van der Aa syndrome and Noonan syndrome. We observed a morphological overlap with the Noonan-like disorder with anagen hair in our patients.