Abnormality of Early White Matter Development in Tuberous Sclerosis Complex and Autism Spectrum Disorder: Longitudinal Analysis of Diffusion Tensor Imaging Measures.

Background: Abnormalities in white matter development may influence development of autism spectrum disorder in tuberous sclerosis complex (TSC). Our goals for this study were as follows: (1) use data from a longitudinal neuroimaging study of tuberous sclerosis complex (TACERN) to develop optimized linear mixed effects models for analyzing longitudinal, repeated diffusion tensor imaging metrics (fractional anisotropy, mean diffusivity) pertaining to select white matter tracts, in relation to positive Autism Diagnostic Observation Schedule-Second Edition classification at 36 months, and (2) perform an exploratory analysis using optimized models applied to all white matter tracts from these data. Methods: Eligible participants (3-12 months) underwent brain magnetic resonance imaging (MRI) at repeated time points from ages 3 to 36 months. Positive Autism Diagnostic Observation Schedule-Second Edition classification at 36 months was used. Linear mixed effects models were fine-tuned separately for fractional anisotropy values (using fractional anisotropy corpus callosum as test outcome) and mean diffusivity values (using mean diffusivity right posterior limb internal capsule as test outcome). Fixed effects included participant age, within-participant longitudinal age, and autism spectrum disorder diagnosis. Results: Analysis included data from n = 78. After selecting separate optimal models for fractional anisotropy and mean diffusivity values, we applied these models to fractional anisotropy and mean diffusivity of all 27 white matter tracts. Fractional anisotropy corpus callosum was related to positive Autism Diagnostic Observation Schedule-Second Edition classification (coefficient = 0.0093, P = .0612), and mean diffusivity right inferior cerebellar peduncle was related to positive Autism Diagnostic Observation Schedule-Second Edition classification (coefficient = -0.00002071, P = .0445), though these findings were not statistically significant after multiple comparisons correction. Conclusion: These optimized linear mixed effects models possibly implicate corpus callosum and cerebellar pathology in development of autism spectrum disorder in tuberous sclerosis complex, but future studies are needed to replicate these findings and explore contributors of heterogeneity in these models.

Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders.

Variant detection from long-read genome sequencing (lrGS) has proven to be considerably more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences "HiFi" technology on 96 short-read-negative probands with rare disease that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, eight of which (8/96, 8.33%) harbored pathogenic or likely pathogenic variants. Newly identified variants were visible in both srGS and lrGS in nine probands (~9.4%) and resulted from changes to interpretation mostly from recent gene-disease association discoveries. Seven cases included variants that were only interpretable in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either: not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Final analysis of potential drug-drug interactions between highly purified cannabidiol and anti-seizure medications in an open-label expanded access program.

OBJECTIVE: The aim of this study was to assess potential drug-drug interactions between highly purified cannabidiol (CBD) and anti-seizure medications (ASMs). METHODS: Our group previously reported that in a sample of adults and children receiving CBD in an open-label expanded access program, there were several ASMs noted to increase in serum levels with increasing doses of CBD. We analyzed if an increased number of observations over time resulted in changes in potential interactions and if potential interactions were associated with time since enrollment, demographics, or the overall rating of adverse effects. RESULTS: In 169 participants (80 adults), with increasing weight-based CBD dose, there were associated increases in serum levels of clobazam and N-desmethylclobazam, free valproate, felbamate, and topiramate in the adult and pediatric arms combined, levetiracetam in the pediatric arm only, and permapanel in the adult arm only. There were no associations noted in these level changes with time since enrollment, biological sex, and adverse events profile scores. SIGNIFICANCE: This study confirms some previously identified interactions with CBD and identifies other potential pharmacokinetic interactions; however, the clinical significance of these observations is likely minor, and there is no effect of time on these findings.

Efficacy of cannabidiol in convulsive and nonconvulsive seizure types associated with treatment-resistant epilepsies in the Expanded Access Program.

The cannabidiol (CBD) Expanded Access Program (EAP), initiated in 2014, provided CBD (Epidiolex) to patients with treatment-resistant epilepsy (TRE). In the final pooled analysis of 892 patients treated through January 2019 (median exposure = 694 days), CBD treatment was associated with a 46%-66% reduction in median monthly total (convulsive plus nonconvulsive) seizure frequency. CBD was well tolerated, and adverse events were consistent with previous findings. We used pooled EAP data to investigate the effectiveness of add-on CBD therapy for individual convulsive seizure types (clonic, tonic, tonic-clonic, atonic, focal to bilateral tonic-clonic), nonconvulsive seizure types (focal with and without impaired consciousness, absence [typical and atypical], myoclonic, myoclonic absence), and epileptic spasms. CBD treatment was associated with a reduction in the frequency of convulsive seizure types (median percentage reduction = 47%-100%), and nonconvulsive seizure types and epileptic spasms (median percentage reduction = 50%-100%) across visit intervals through 144 weeks of treatment. Approximately 50% of patients had ≥50% reduction in convulsive and nonconvulsive seizure types and epileptic spasms at nearly all intervals. These results show a favorable effect of long-term CBD use in patients with TRE, who may experience various convulsive and nonconvulsive seizure types. Future controlled trials are needed to confirm these findings.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Epilepsy Severity Is Associated With Head Circumference and Growth Rate in Infants With Tuberous Sclerosis Complex.

BACKGROUND: Abnormal brain growth in tuberous sclerosis complex (TSC) reflects abnormalities in cellular proliferation and differentiation and results in epilepsy and other neurological manifestations. Head circumference (HC) as a proxy for brain volume may provide an easily tracked clinical measure of brain overgrowth and neurological disease burden. This study investigated the relationship between HC and epilepsy severity in infants with TSC. METHODS: Prospective multicenter observational study of children from birth to three years with TSC. Epilepsy data were collected from clinical history, and HC was collected at study visits at age three, six, nine, 12, 18, 24, and 36 months. Epilepsy severity was classified as no epilepsy, low epilepsy severity (one seizure type and one or two antiepileptic drugs [AEDs]), moderate epilepsy severity (either two to three seizure types and one to two AEDs or one seizure type and more than three AEDs), or high epilepsy severity (two to three seizure types and more than three AEDs). RESULTS: As a group, children with TSC had HCs approximately 1 S.D. above the mean World Health Organization (WHO) reference by age one year and demonstrated more rapid growth than the normal population reference. Males with epilepsy had larger HCs than those without. Compared with the WHO reference population, infants with TSC and no epilepsy or low or moderate epilepsy had an increased early HC growth rate, whereas those with severe epilepsy had an early larger HC but did not have a faster growth rate. CONCLUSIONS: Infants and young children with TSC have larger HCs than typical growth norms and have differing rates of head growth depending on the severity of epilepsy.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

Purpose: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains around 50%, suggesting some clinically relevant rare variants may be missed by standard analyses. Here we analyze "poison exons" (PEs) which, while often absent from standard gene annotations, are alternative exons whose inclusion results in a premature termination codon. Variants that alter PE inclusion can lead to loss-of-function and may be highly penetrant contributors to disease. Methods: We curated published RNA-seq data from developing mouse cortex to define 1,937 PE regions conserved between humans and mice and potentially relevant to NDDs. We then analyzed variants found by genome sequencing in multiple NDD cohorts. Results: Across 2,999 probands, we found six clinically relevant variants in PE regions that were previously overlooked. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family ( SCN1A, SCN2A , and SCN8A ), associated with epilepsies. One variant is in SNRPB , associated with Cerebrocostomandibular Syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and were observed in probands with features consistent with those reported for the associated gene. Conclusion: With only a minimal increase in variant analysis burden (most probands had zero or one candidate PE variants in a known NDD gene, with an average of 0.77 per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Tubers Affecting the Fusiform Face Area Are Associated with Autism Diagnosis.

OBJECTIVE: Tuberous sclerosis complex (TSC) is associated with focal brain "tubers" and a high incidence of autism spectrum disorder (ASD). The location of brain tubers associated with autism may provide insight into the neuroanatomical substrate of ASD symptoms. METHODS: We delineated tuber locations for 115 TSC participants with ASD (n = 31) and without ASD (n = 84) from the Tuberous Sclerosis Complex Autism Center of Excellence Research Network. We tested for associations between ASD diagnosis and tuber burden within the whole brain, specific lobes, and at 8 regions of interest derived from the ASD neuroimaging literature, including the anterior cingulate, orbitofrontal and posterior parietal cortices, inferior frontal and fusiform gyri, superior temporal sulcus, amygdala, and supplemental motor area. Next, we performed an unbiased data-driven voxelwise lesion symptom mapping (VLSM) analysis. Finally, we calculated the risk of ASD associated with positive findings from the above analyses. RESULTS: There were no significant ASD-related differences in tuber burden across the whole brain, within specific lobes, or within a priori regions derived from the ASD literature. However, using VLSM analysis, we found that tubers involving the right fusiform face area (FFA) were associated with a 3.7-fold increased risk of developing ASD. INTERPRETATION: Although TSC is a rare cause of ASD, there is a strong association between tuber involvement of the right FFA and ASD diagnosis. This highlights a potentially causative mechanism for developing autism in TSC that may guide research into ASD symptoms more generally. ANN NEUROL 2023;93:577-590.

Limited utility of structural MRI to identify the epileptogenic zone in young children with tuberous sclerosis.

BACKGROUND AND PURPOSE: The success of epilepsy surgery in children with tuberous sclerosis complex (TSC) hinges on identification of the epileptogenic zone (EZ). We studied structural MRI markers of epileptogenic lesions in young children with TSC. METHODS: We included 26 children with TSC who underwent epilepsy surgery before the age of 3 years at five sites, with 12 months or more follow-up. Two neuroradiologists, blinded to surgical outcome data, reviewed 10 candidate lesions on preoperative MRI for characteristics of the tuber (large affected area, calcification, cyst-like properties) and of focal cortical dysplasia (FCD) features (cortical malformation, gray-white matter junction blurring, transmantle sign). They selected lesions suspect for the EZ based on structural MRI, and reselected after unblinding to seizure onset location on electroencephalography (EEG). RESULTS: None of the tuber characteristics and FCD features were distinctive for the EZ, indicated by resected lesions in seizure-free children. With structural MRI alone, the EZ was identified out of 10 lesions in 31%, and with addition of EEG data, this increased to 48%. However, rates of identification of resected lesions in non-seizure-free children were similar. Across 251 lesions, interrater agreement was moderate for large size (κ = .60), and fair (κ = .24) for all other features. CONCLUSIONS: In young children with TSC, the utility of structural MRI features is limited in the identification of the epileptogenic tuber, but improves when combined with EEG data.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Disruption of MeCP2-TCF20 complex underlies distinct neurodevelopmental disorders.

MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function.

Long-term cannabidiol treatment for seizures in patients with tuberous sclerosis complex: An open-label extension trial.

OBJECTIVE: To evaluate the long-term safety and efficacy of add-on cannabidiol (CBD) in patients with seizures associated with tuberous sclerosis complex (TSC) in the open-label extension (OLE) of the randomized, placebo-controlled phase 3 trial GWPCARE6 (NCT02544763). Results of an interim (February 2019 data cut) analysis are reported. METHODS: Patients who completed the randomized trial enrolled to receive CBD (Epidiolex® in the United States; Epidyolex® in the EU; 100 mg/mL oral solution). The initial target dose was 25 mg/kg/day, which, based on response and tolerability, could be decreased or increased up to 50 mg/kg/day. The primary end point was safety. Key secondary end points included percentage reduction in TSC-associated (countable focal and generalized) seizures, responder rates, and Subject/Caregiver Global Impression of Change (S/CGIC). RESULTS: Of 201 patients who completed the randomized phase, 199 (99%) entered the OLE. Mean age was 13 years (range, 1-57). At the time of analysis, 5% of patients had completed treatment, 20% had withdrawn, and 75% were ongoing. One-year retention rate was 79%. Median treatment time was 267 days (range, 18-910) at a 27 mg/kg/day mean modal dose. Most patients (92%) had an adverse event (AE). Most common AEs were diarrhea (42%), seizure (22%), and decreased appetite (20%). AEs led to permanent discontinuation in 6% of patients. There was one death that was deemed treatment unrelated by the investigator. Elevated liver transaminases occurred in 17 patients (9%) patients; 12 were taking valproate. Median percentage reductions in seizure frequency (12-week windows across 48 weeks) were 54%-68%. Seizure responder rates (≥50%, ≥75%, 100% reduction) were 53%-61%, 29%-45%, and 6%-11% across 12-week windows for 48 weeks. Improvement on the S/CGIC scale was reported by 87% of patients/caregivers at 26 weeks. SIGNIFICANCE: In patients with TSC, long-term add-on CBD treatment was well tolerated and sustainably reduced seizures through 48 weeks, with most patients/caregivers reporting global improvement.

Updated International Tuberous Sclerosis Complex Diagnostic Criteria and Surveillance and Management Recommendations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease affecting multiple body systems with wide variability in presentation. In 2013, Pediatric Neurology published articles outlining updated diagnostic criteria and recommendations for surveillance and management of disease manifestations. Advances in knowledge and approvals of new therapies necessitated a revision of those criteria and recommendations. METHODS: Chairs and working group cochairs from the 2012 International TSC Consensus Group were invited to meet face-to-face over two days at the 2018 World TSC Conference on July 25 and 26 in Dallas, TX, USA. Before the meeting, working group cochairs worked with group members via e-mail and telephone to (1) review TSC literature since the 2013 publication, (2) confirm or amend prior recommendations, and (3) provide new recommendations as required. RESULTS: Only two changes were made to clinical diagnostic criteria reported in 2013: "multiple cortical tubers and/or radial migration lines" replaced the more general term "cortical dysplasias," and sclerotic bone lesions were reinstated as a minor criterion. Genetic diagnostic criteria were reaffirmed, including highlighting recent findings that some individuals with TSC are genetically mosaic for variants in TSC1 or TSC2. Changes to surveillance and management criteria largely reflected increased emphasis on early screening for electroencephalographic abnormalities, enhanced surveillance and management of TSC-associated neuropsychiatric disorders, and new medication approvals. CONCLUSIONS: Updated TSC diagnostic criteria and surveillance and management recommendations presented here should provide an improved framework for optimal care of those living with TSC and their families.

Epilepsy Is Heterogeneous in Early-Life Tuberous Sclerosis Complex.

BACKGROUND: Epilepsy in tuberous sclerosis complex (TSC) typically presents with early onset, multiple seizure types, and intractability. However, variability is observed among individuals. Here, detailed individual data on seizure characteristics collected prospectively during early life were used to define epilepsy profiles in this population. METHODS: Children aged zero to 36 months were followed longitudinally. Caregivers kept daily seizure diaries, including onset and daily counts for each seizure type. Patients with >70% seizure diary completion and >365 diary days were included. Developmental outcomes at 36 months were compared between subgroups. RESULTS: Epilepsy was seen in 124 of 156 (79%) participants. Seizure onset occurred from zero to 29.5 months; 93% had onset before age 12 months. Focal seizures and epileptic spasms were most common. Number of seizures (for median 897 days) ranged from 1 to 9128. Hierarchical clustering based on six metrics of seizure burden (age of onset, total seizures, ratio of seizure days to nonseizure days, seizures per seizure day, and worst seven- and 30-day stretches) revealed two distinct groups with broadly favorable and unfavorable epilepsy profiles. Subpopulations within each group showed clinically meaningful differences in seizure burden. Groups with higher seizure burden had worse developmental outcomes at 36 months. CONCLUSIONS: Although epilepsy is highly prevalent in TSC, not all young children with TSC have the same epilepsy profile. At least two phenotypic subpopulations are discernible based on seizure burden. Early and aggressive treatments for epilepsy in TSC may be best leveraged by targeting specific subgroups based on phenotype severity.

Pharmacogenetic Predictors of Cannabidiol Response and Tolerability in Treatment-Resistant Epilepsy.

In patients with treatment-resistant epilepsy (TRE), cannabidiol (CBD) produces variable improvement in seizure control. Patients in the University of Alabama at Birmingham CBD Expanded Access Program (EAP) were enrolled in the genomic study and genotyped using the Affymetrix Drug Metabolizing Enzymes and Transporters plus array. Associations between variants and CBD response (≥50% seizure reduction) and tolerability (diarrhea, sedation, and abnormal liver function) was evaluated under dominant and recessive models. Expression quantitative trait loci (eQTL) influencing potential CBD targets was evaluated in the UK Brain Expression Consortium data set (Braineac), and genetic co-expression examined. Of 169 EAP patients, 112 (54.5% pediatric and 50.0% female) were included in the genetic analyses. Patients with AOX1 rs6729738 CC (aldehyde oxidase; odds ratio (OR) 6.69, 95% confidence interval (CI) 2.19-20.41, P = 0.001) or ABP1 rs12539 (diamine oxidase; OR 3.96, 95% CI 1.62-9.73, P = 0.002) were more likely to respond. Conversely, patients with SLC15A1 rs1339067 TT had lower odds of response (OR 0.06, 95% CI 0.01-0.56, P = 0.001). ABCC5 rs3749442 was associated with lower likelihood of response and abnormal liver function tests, and higher likelihood of sedation. The eQTL revealed that rs1339067 decreased GPR18 expression (endocannabinoid receptor) in white matter (P = 5.6 × 10-3 ), and rs3749442 decreased hippocampal HTR3E expression (serotonin 5-HT3E ; P = 8.5 × 10-5 ). Furthermore, 75% of genes associated with lower likelihood of response were co-expressed. Pharmacogenetic variation is associated with CBD response and influences expression of CBD targets in TRE. Implicated pathways, including cholesterol metabolism and glutathione conjugation, demonstrate potential interactions between CBD and common medications (e.g., statins and acetaminophen) that may require closer monitoring. These results highlight the role of pharmacogenes in fundamental biologic processes and potential genetic underpinnings of treatment-resistance.

Long-read genome sequencing for the molecular diagnosis of neurodevelopmental disorders.

Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches. Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads. We identified variants and created de novo assemblies in each trio, with global metrics indicating these datasets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic. The breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs, support the hypothesis that long-read genome sequencing can substantially improve rare disease genetic discovery rates.

Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy.

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism.

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.