A deep intronic DLG4 variant resulting in DLG4-related synaptopathy.

The rare autosomal dominant brain disorder DLG4-related synaptopathy is caused by de novo variants in DLG4 (encoding PSD-95), the majority of which are predicted to be protein-truncating. In addition to splice site variants, a number of synonymous and missense DLG4 variants are predicted to exert their effect through altered RNA splicing, although the pathogenicity of these variants is uncertain without functional RNA studies. Here, we describe a young boy with a deep intronic DLG4 variant (c.2105+235C>T) identified using whole genome sequencing. By using reverse-transcription PCR on RNA derived from peripheral blood, we demonstrate that DLG4 mRNA expression is detectable in blood and the deep intronic variant gives rise to two alternative DLG4 transcripts, one of which includes a pseudoexon. Both alternative transcripts are out-of-frame and predicted to result in protein-truncation, thereby establishing the genetic diagnosis for the proband. This adds to the evidence concerning the pathogenic potential of deep intronic variants and underlines the importance of functional studies, even in cases where reported tissue-specific gene expression might suggest otherwise.

The phenotypic and genotypic spectrum of individuals with mono- or biallelic ANK3 variants.

ANK3 encodes ankyrin-G, a protein involved in neuronal development and signaling. Alternative splicing gives rise to three ankyrin-G isoforms comprising different domains with distinct expression patterns. Mono- or biallelic ANK3 variants are associated with non-specific syndromic intellectual disability in 14 individuals (seven with monoallelic and seven with biallelic variants). In this study, we describe the clinical features of 13 additional individuals and review the data on a total of 27 individuals (16 individuals with monoallelic and 11 with biallelic ANK3 variants) and demonstrate that the phenotype for biallelic variants is more severe. The phenotypic features include language delay (92%), autism spectrum disorder (76%), intellectual disability (78%), hypotonia (65%), motor delay (68%), attention deficit disorder (ADD) or attention deficit hyperactivity disorder (ADHD) (57%), sleep disturbances (50%), aggressivity/self-injury (37.5%), and epilepsy (35%). A notable phenotypic difference was presence of ataxia in three individuals with biallelic variants, but in none of the individuals with monoallelic variants. While the majority of the monoallelic variants are predicted to result in a truncated protein, biallelic variants are almost exclusively missense. Moreover, mono- and biallelic variants appear to be localized differently across the three different ankyrin-G isoforms, suggesting isoform-specific pathological mechanisms.

Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

Biallelic variants in ZNF142 lead to a syndromic neurodevelopmental disorder.

Biallelic variants of the gene encoding for the zinc-finger protein 142 (ZNF142) have recently been associated with intellectual disability (ID), speech impairment, seizures, and movement disorders in nine individuals from five families. In this study, we obtained phenotype and genotype information of 26 further individuals from 16 families. Among the 27 different ZNF142 variants identified in the total of 35 individuals only four were missense. Missense variants may give a milder phenotype by changing the local structure of ZF motifs as suggested by protein modeling; but this correlation should be validated in larger cohorts and pathogenicity of the missense variants should be investigated with functional studies. Clinical features of the 35 individuals suggest that biallelic ZNF142 variants lead to a syndromic neurodevelopmental disorder with mild to moderate ID, varying degrees of delay in language and gross motor development, early onset seizures, hypotonia, behavioral features, movement disorders, and facial dysmorphism. The differences in symptom frequencies observed in the unpublished individuals compared to those of published, and recognition of previously underemphasized facial features are likely to be due to the small sizes of the previous cohorts, which underlines the importance of larger cohorts for the phenotype descriptions of rare genetic disorders.

Neurodevelopmental Disorders Associated with PSD-95 and Its Interaction Partners.

The postsynaptic density (PSD) is a massive protein complex, critical for synaptic strength and plasticity in excitatory neurons. Here, the scaffolding protein PSD-95 plays a crucial role as it organizes key PSD components essential for synaptic signaling, development, and survival. Recently, variants in DLG4 encoding PSD-95 were found to cause a neurodevelopmental disorder with a variety of clinical features including intellectual disability, developmental delay, and epilepsy. Genetic variants in several of the interaction partners of PSD-95 are associated with similar phenotypes, suggesting that deficient PSD-95 may affect the interaction partners, explaining the overlapping symptoms. Here, we review the transmembrane interaction partners of PSD-95 and their association with neurodevelopmental disorders. We assess how the structural changes induced by DLG4 missense variants may disrupt or alter such protein-protein interactions, and we argue that the pathological effect of DLG4 variants is, at least partly, exerted indirectly through interaction partners of PSD-95. This review presents a direction for functional studies to elucidate the pathogenic mechanism of deficient PSD-95, providing clues for therapeutic strategies.

Involvement of Mitochondrial Dysfunction in FOXG1 Syndrome.

FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.

Candidate Genes and Pathways Associated with Gilles de la Tourette Syndrome-Where Are We?

Gilles de la Tourette syndrome (GTS) is a childhood-onset neurodevelopmental and -psychiatric tic-disorder of complex etiology which is often comorbid with obsessive-compulsive disorder (OCD) and/or attention deficit hyperactivity disorder (ADHD). Twin and family studies of GTS individuals have shown a high level of heritability suggesting, that genetic risk factors play an important role in disease etiology. However, the identification of major GTS susceptibility genes has been challenging, presumably due to the complex interplay between several genetic factors and environmental influences, low penetrance of each individual factor, genetic diversity in populations, and the presence of comorbid disorders. To understand the genetic components of GTS etiopathology, we conducted an extensive review of the literature, compiling the candidate susceptibility genes identified through various genetic approaches. Even though several strong candidate genes have hitherto been identified, none of these have turned out to be major susceptibility genes yet.

EWAS of Monozygotic Twins Implicate a Role of mTOR Pathway in Pathogenesis of Tic Spectrum Disorder.

Tic spectrum disorder (TSD) is an umbrella term which includes Gilles de la Tourette syndrome (GTS) and chronic tic disorder (CTD). They are considered highly heritable, yet the genetic components remain largely unknown. In this study we aimed to investigate disease-associated DNA methylation differences to identify genes and pathways which may be implicated in TSD aetiology. For this purpose, we performed an exploratory analysis of the genome-wide DNA methylation patterns in whole blood samples of 16 monozygotic twin pairs, of which eight were discordant and six concordant for TSD, while two pairs were asymptomatic. Although no sites reached genome-wide significance, we identified several sites and regions with a suggestive significance, which were located within or in the vicinity of genes with biological functions associated with neuropsychiatric disorders. The two top genes identified (TSC1 and CRYZ/TYW3) and the enriched pathways and components (phosphoinosides and PTEN pathways, and insulin receptor substrate binding) are related to, or have been associated with, the PI3K/AKT/mTOR pathway. Genes in this pathway have previously been associated with GTS, and mTOR signalling has been implicated in a range of neuropsychiatric disorders. It is thus possible that altered mTOR signalling plays a role in the complex pathogenesis of TSD.

Elevated Expression of SLC6A4 Encoding the Serotonin Transporter (SERT) in Gilles de la Tourette Syndrome.

Gilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder characterized by motor and vocal tics. Most of the GTS individuals have comorbid diagnoses, of which obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) are the most common. Several neurotransmitter systems have been implicated in disease pathogenesis, and amongst these, the dopaminergic and the serotonergic pathways are the most widely studied. In this study, we aimed to investigate whether the serotonin transporter (SERT) gene (SLC6A4) was differentially expressed among GTS individuals compared to healthy controls, and whether DNA variants (the SERT-linked polymorphic region 5-HTTLPR, together with the associated rs25531 and rs25532 variants, and the rare Ile425Val variant) or promoter methylation of SLC6A4 were associated with gene expression levels or with the presence of OCD as comorbidity. We observed that SLC6A4 expression is upregulated in GTS individuals compared to controls. Although no specific genotype, allele or haplotype was overrepresented in GTS individuals compared to controls, we observed that the LAC/LAC genotype of the 5-HTTLPR/rs25531/rs25532 three-locus haplotype was associated with higher SLC6A4 mRNA expression levels in GTS individuals, but not in the control group.