VAMP2 Gene-Related Neurodevelopmental Disorder: A Differential Diagnosis for Rett/Angelman-Type Spectrum of Disorders

Introduction

Rapid communication across neuronal synapses depends on activity-dependent and coordinated neurotransmitter release [Südhof and Rothman, 2009]. This is achieved via fusion of synaptic vesicles with the presynaptic membrane and subsequent neurotransmitter exocytosis [Jahn and Fasshauer, 2012]. This process is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins [Salpietro et al., 2019]: vesicle-associated membrane protein 2 (VAMP2, also called synaptobrevin-2) on the vesicle membrane and syntaxin1A (STX1A) and synaptosomal-associated protein 25 (SNAP25) on the plasma membrane [Fasshauer et al., 1997; Salpietro et al., 2019]. STX1A and SNAP25 form a binary complex and assemble with VAMP2 to form a SNARE complex which “zippers” the vesicular and synaptic membranes together, resulting in fusion of the lipid bilayers [Gao et al., 2012]. These proteins are highly conserved, suggesting they have an important biological function in synaptic transmission [Fasshauer et al., 1998].

The SNARE complex also interacts with regulatory proteins including Munc13, Munc18, and complexins, and the main Ca²⁺ sensor, synaptotagmin-1 [Rizo and Rosenmund, 2008; Salpietro et al., 2019]. Upon membrane depolarisation resulting from an action potential, voltage-gated Ca²⁺ channels open, resulting in an influx of Ca²⁺ which is detected by synaptotagmin-1 [Simmons et al., 2020]. Synaptotagmin-1 signals this rise in concentration to the SNARE complex, which then initiates membrane fusion [Simmons et al., 2020]. It has been suggested that a single SNARE complex might be sufficient to initiate membrane fusion, but the use of multiple complexes facilitates rapid and synchronised neurotransmitter release [Morhmann and Sørensen, 2012].

VAMP2 is encoded by the VAMP2 gene, which is located on chromosome 17p13.1 and consists of six exons [Costa et al., 2022]. Previous in vitro studies have demonstrated that pathogenic variants affecting its SNARE motif (sequence of the protein with a tendency to form coiled coils [Rizo and Rosenmund, 2008]) can interfere with liposomal fusion and promotion of plasma membrane “zippering” via Munc18 [Hernandez et al., 2012; Shen et al., 2015]. Experiments in mice have also demonstrated that homozygous deletion of VAMP2 results in lethality at birth, impairment of neurotransmitter release, and delayed synaptic vesicle endocytosis [Schoch et al., 2001; Deak et al., 2004; Monteggia et al., 2019]. Loss of one VAMP2 allele in mice does not appear to cause premature mortality nor impairments in learning or memory, and these mice seem to have improved coordination compared to wild-type controls [Schoch et al., 2001; Monteggia et al., 2019]. In humans, Salpietro et al. [2019] demonstrated that pathogenic variants in the region coding for the VAMP2 SNARE motif result in impairment of vesicle fusion. All 5 patients in this study had heterozygous de novo pathogenic variants in the VAMP2 gene and all presented with axial hypotonia (present since birth), intellectual disability, and features of autism spectrum disorder. There have been a further 6 patients described in the literature with pathogenic variants affecting the VAMP2 gene. Sunaga et al. [2020] report one patient with a pathogenic variant in the VAMP2 gene SNARE motif region presenting with hypotonia, infantile spasms, poor visual fixation, and absence of purposeful hand movements. Simmons and colleagues [2020] report 5 patients, three with pathogenic variants in the region coding for the SNARE motif, one in the N-terminal domain, and one in the transmembrane domain. All 5 patients presented with global developmental delay, intellectual disability, and features of autism spectrum disorder.

Here, we describe a novel de novo heterozygous missense variant in the VAMP2 gene in a patient presenting with early onset hypotonia, global developmental delay, speech delay, microcephaly, and stereotypic hand movements. This case adds to the genotype-phenotype correlation in VAMP2 gene-related neurodevelopmental disorder and identifies this condition as an important differential diagnosis of Rett/Angelman-type spectrum of disorders.

Case Report

A 15-months-old girl presented with early onset hypotonia, global developmental delay, speech delay, microcephaly, nystagmus, strabismus, and stereotypic hand movements (flapping and clapping). She was born after an uneventful pregnancy via elective caesarean section at 38 weeks of gestation. Her birth weight was 2.92 kg (−2.68 SD), length 0 SD, and occipitofrontal circumference −2.01 SD. She was the second child of a healthy non-consanguineous Caucasian couple. She had no difficulties with feeding in the neonatal period and smiled by 6 weeks of age. She was felt to be hypotonic from birth, and significant global developmental delay emerged over the first few months of life. She first sat independently at age of 12 months and started babbling at age of 18 months. At age of 21 months, she remained hypotonic and was able to sit steadily and roll over but not cruise. She could say a few single words with meaning, and her understanding was reported to be good by her parents. By 2 years and 9 months of age, she was able to crawl and stand against the furniture and had a few two-word phrases. Frequent teeth grinding was also noted.

By age of 5 years, she was able to walk with support though had joint laxity necessitating orthopaedic footwear. Her gait was slightly ataxic with a broad base. She had deep-set eyes, a prominent nose, small ears, and strabismus (shown in Fig. 1). She had significant learning difficulties and attended a school for children with special needs. She had several stereotypies described as hand flapping, hand wringing, and tongue protrusions. She had a happy and affectionate demeanour. She received input from physiotherapy, occupational therapy, and speech and language therapy. By age of 7 years, she was able to walk unaided and run. She was only able to speak single words but could anticipate events during the day and sing the tunes of nursery rhymes. Stereotypical hand movements persisted, and eye movements were jerky.

By age of 9 years, she was able to walk up and down stairs with support, and walk around the house, but required a wheelchair for longer distances. She had a short attention span and tended not to play with other children. Around this age, she developed significant constipation, a fragmented sleep disorder, and challenging behaviour. She had episodic agitation, aggression (spitting, biting, scratching, and hitting) towards others and herself. By age of 16 years, she had developed, over a 2-years period, a severe scoliosis of the thoracolumbar spine necessitating surgical intervention and experienced episodes of head-hitting. She was prescribed quetiapine to achieve satisfactory sleep. She was able to feed herself single items of finger food and her speech remained mainly limited to single words, with occasional short sentences. She was able to use the Picture Exchange Communication System to make simple decisions.

Initially, the working diagnoses were Angelman syndrome and Rett syndrome in view of the presenting features of global developmental delay, hypotonia, stereotypical movements, speech delay, and microcephaly. Routine karyotype, metabolic investigations, testing for myotonic dystrophy, spinal muscular atrophy, Prader-Willi syndrome, MECP2 gene testing and duplication-deletion analysis, chromosome 15 Angelman syndrome critical region analysis, UBE3A gene analysis, CREBBP gene analysis for Rubinstein-Taybi syndrome (given occipitofrontal circumference −2.01 SD and prominent nose), and FOXG1 gene analysis were all unremarkable. Array comparative genomic hybridisation identified a 10q23.1 deletion which was inherited from the proband’s mother and therefore unlikely to be significant as there were no maternal phenotypic abnormalities. Cranial magnetic resonance imaging undertaken at both 13 months of age and 4 years of age was normal. Magnetic resonance imaging of the whole spine at 15 years of age demonstrated a significant thoracolumbar scoliosis with no appreciable cause evident. The proband was enrolled in the Deciphering Developmental Disorders (DDD) study at age of 5 years and the 100,000 Genomes Project at age of 9 years.

Analysis of the 100,000 Genomes Project whole genome sequencing data identified a novel de novo heterozygous missense variant in the VAMP2 gene: NM_014232.3 c.197G>C (p.Arg66Pro). Following this, the variant was also identified as a candidate variant for review by the DDD study. The variant was classified as likely pathogenic according to the American College of Medical Genetics and Genomics criteria [Richards et al., 2015] and Association for Clinical Genomic Service Variant Interpretation guidelines [Ellard et al., 2020]. It is not present in the gnomAD population database, in silico computational tools predict a damaging effect (PolyPhen-2, SIFT), and the variant is present within a region of the gene that is highly constrained for missense variation. The variant also occurred de novo in the proband, which was considered a strong piece of evidence given the consistency of the proband’s phenotype with others reported previously by Salpietro et al. [2019]. Figure 2 shows the VAMP2 gene and protein structure and demonstrates the location of the variants in the patients previously reported by Salpietro et al. [2019], Sunaga et al. [2020], and Simmons et al. [2020] and this patient.

Discussion

We present the twelfth reported case of VAMP2 gene-related neurodevelopmental disorder, an emerging rare genetic condition, due to a novel de novo heterozygous missense variant in the SNARE motif region of the VAMP2 gene. The patient presented with early onset hypotonia, global developmental delay, learning difficulties, microcephaly, nystagmus, strabismus, and stereotypies and later developed a sleep disorder, challenging behaviour with self-injury, and scoliosis. This case identifies VAMP2 gene-related neurodevelopmental disorder as an important differential diagnosis of Rett/Angelman-type spectrum of disorders.

VAMP2 is highly expressed in the putamen and frontal lobes of the brain [Salpietro et al., 2019], regions involved in language, motor control, learning, and executive function [Chayer and Freedman, 2001; Ghandili and Munakomi, 2022]. It follows therefore that pathogenic variants in the VAMP2 gene might result in deficits in these brain functions, as exhibited by patients reported to date in the literature presenting with developmental delay and intellectual disability [Salpietro et al., 2019; Sunaga et al., 2020; Simmons et al., 2020]. A reduction in VAMP2 gene expression in the hippocampus and entorhinal cortex has also been demonstrated in patients with Alzheimer’s disease, with levels correlating with the degree of cognitive dysfunction [Sze et al., 2000]. Downregulation of VAMP2 gene expression has also been implicated in patients with comorbid epilepsy and ADHD [Xi et al., 2020].

To date, only 11 different variants in the VAMP2 gene have been previously reported in 11 patients with features consistent with VAMP2 gene-related neurodevelopmental disorder (Table 1) [Salpietro et al., 2019; Sunaga et al., 2020; Simmons et al., 2020]. The main clinical features include global developmental delay, intellectual disability, features of autism spectrum disorder, speech delay, behavioural and psychological abnormalities (including self-injury, hyperactivity, and impulsivity), and hypotonia. Where reported, all patients had either repetitive stereotypical hand movements, a typical feature of Rett syndrome [Carter et al., 2010], or an absence of purposeful hand movements [Salpietro et al., 2019; Sunaga et al., 2020].

All missense variants reported are present within the SNARE domain and are predicted to cause a dominant-negative effect, based on previous computational and experimental evidence [Grote and Kelly, 1996; Koo et al., 2011; Simmons et al., 2020]. Missense variants appear to be associated with a more severe phenotype, including seizures and severe intellectual disability (patients 3, 4, and 7–10 in Table 1), whereas loss-of-function variants resulting in haploinsufficiency tend to result in a milder phenotype, including an age-appropriate level of independence, no seizures, ability to speak at least several words, and ability to walk (patients 2, 5, and 6 in Table 1). The latter supposition is based on a small sample size, as there are only three predicted loss-of-function variants reported in the literature. However, there are several diseases where dominant-negative mechanisms are known to result in a more severe phenotype, for example, osteogenesis imperfecta. It is predicted that haploinsufficiency operates as a different disease mechanism for VAMP2 gene-related neurodevelopmental disorder compared to the dominant-negative effect of reported missense variants. Patients 11 and 12 both have single amino acid deletions and exhibit a similar mild phenotype to compared patients 2, 5, and 6; the disease mechanism is unclear for these in-frame deletions.

The variant in patient 8 (c.223T>C, p.Ser75Pro) was shown to have a dominant-negative effect when tested in vitro under Munc18 conditions, whereas the variant in patient 9 (c.233A>C, p.Glu78Ala) had no impact on the SNARE assembly process under the same conditions [Salpietro et al., 2019]. This patient’s phenotype may be explained by defective interactions with other regulatory proteins which were not present in the test assay [Salpietro et al., 2019]. The variant in patient 10 could not be purified satisfactorily for in vitro testing.

The present patient has a novel de novo missense variant in the VAMP2 gene region which codes for the SNARE domain: NM_014232.3 c.197G>C (p.Arg66Pro). Her clinical manifestations were moderate, with some features (limited walking and complete dependence in ADLs) consistent with previously reported more severe phenotypes associated with variants causing a dominant-negative effect and others (moderate intellectual disability, many spoken words, and no seizures) consistent with loss-of-function variants resulting in a milder phenotype. She also had microcephaly and developed scoliosis, which have not been reported previously in VAMP2 gene-related neurodevelopmental disorder. In the context of a phenotype that is moderate compared to previous missense variants reported, it is possible that this missense variant is exhibiting its effect either through haploinsufficiency, a dominant-negative effect, or another mechanism or combination of mechanisms, and further study is required to establish this.

This case also identifies VAMP2 gene-related neurodevelopmental disorder as an important differential diagnosis of Rett/Angelman-type spectrum of disorders. Rett syndrome is a severe neurodevelopmental disorder in which girls typically present with normal early development followed by a period of stagnation and regression of their skills. There is typically a decline in head growth, loss of purposeful hand movements with development of stereotypical movements such as wringing and clapping, global developmental delay, loss of speech, and later other features including scoliosis [Neul et al., 2010]. The present patient had some features of Rett syndrome (stereotypical hand movements, global developmental delay, poor speech, microcephaly, and later scoliosis) but did not have the cardinal feature of a regression in her hand or language skills; rather, her development was delayed from the first few months of life.

Like Rett syndrome, children with Angelman syndrome appear normal at birth, but usually feeding difficulties, hypotonia, and psychomotor delay emerge in the first 6 months of life [Buiting et al., 2016]. Children with Angelman syndrome continue to develop with severe developmental delay including absent speech, and typically there are characteristic behavioural features such as regular outbursts of laughter with hand flapping movements [Buiting et al., 2016]. In addition, microcephaly usually develops by the age of 3 years and most children have seizures and/or EEG abnormalities and ataxia [Buiting et al., 2016]. The present patient did not fit the typical clinical picture of Angelman syndrome, but she did have several of the consistent clinical features (developmental delay, movement disorder, behavioural features, speech impairment, and microcephaly). Patients presenting with features of either Rett syndrome or Angelman syndrome, in whom genetic testing is not suggestive, should be evaluated for variants in the VAMP2 gene, given the significant overlap in clinical presentation of these disorders. Expanding the range of tests offered to patients presenting with global developmental delay and intellectual disability, who do not fit clearly into a specific disease phenotype, may help reduce the often lengthy delay in diagnosis faced by patients with neurodevelopmental disorders [Harripaul et al., 2017].

Further case reports and functional studies are required to broaden the genotype-phenotype correlation in VAMP2 gene-related neurodevelopmental disorder. Specifically, further research is required to understand the impact of variants resulting in a dominant-negative effect versus haploinsufficiency, on the severity of the clinical presentation. This may also aid in the development of novel therapies aimed at enhancing neurotransmission. Simmons and colleagues [2020] previously trialled 4-aminopyridine, a potassium channel blocker, in a patient with a de novo stop-gain variant in the VAMP2 gene with an improvement in cognition, behaviour, and emotional regulation. Further clinical trials of this treatment may be helpful.

In conclusion, VAMP2 gene-related neurodevelopmental disorder is a newly emerging, rare genetic condition with a spectrum of severity and features which overlap with both Rett syndrome and Angelman syndrome. Expanding genotype-phenotype correlations, and targeted genetic testing, may accelerate diagnosis and in some cases may expedite access to targeted therapies in the future.

Acknowledgments

We thank the patient and her family for their helpful contributions and written consent to complete this work. We also thank Lucy Lancashire for her invaluable administrative support.

This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Ltd. (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK, and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support.

Finally, we thank Dr Julia Foreman (DECIPHER project manager) and the DDD research team. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund (Grant No. HICF-1009-003), a parallel funding partnership between Wellcome and the Department of Health, and the Wellcome Sanger Institute (Grant No. WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of Wellcome or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.

Statement of Ethics

Ethical approval was not required for this study in accordance with local/national guidelines. Written informed consent was obtained from the patient’s parents for publication of the details of their medical case and clinical photographs.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

There was no funding for this case report.

Author Contributions

Swati Naik devised the main conceptual ideas. Danielle Bogue wrote the manuscript. Swati Naik, Gavin Ryan, and Evangeline Wassmer supervised and reviewed the manuscript. All authors approved the final version of the manuscript.

Funding Statement

There was no funding for this case report.

Data Availability Statement

The data that support the findings of this study are openly available in DECIPHER at deciphergenomics.org, reference number [DDD-BWH-264846].