Charlotte Wilhelmina Ockeloen

Variants in BRSK2 , encoding brain specific kinase-2, have recently been associated with an autosomal dominant neurodevelopmental disorder (NDD). We have assembled 52 cases with heterozygous BRSK2 variants and variable neurodevelopmental phenotypes with frequent neuropsychiatric and behavioral symptoms. The variant spectrum included 15 different truncating variants, seven (potential) splice variants, three structural variants, and 12 different missense variants. Of the missense variants, seven were in the kinase domain, and the others in the UBA and the KA1 domain or outside domains. Variants occurred de novo in 19 cases and were inherited in 18. We utilized Drosophila melanogaster as a model and assessed viability and performed climbing and bang sensitivity assays upon knockdown of the fly orthologue sff or upon overexpression of wildtype or mutant human BRSK2 . Pan-neuronal knockdown of sff resulted in impaired locomotor behavior and seizure susceptibility. Ubiquitous or pan-neuronal overexpression of human wildtype BRSK2 in Drosophila resulted in lethality or locomotor impairment, respectively, indicating toxicity. Overexpressing mutant BRSK2 did not or incompletely affect viability and locomotor behavior for six of seven tested kinase domain missense variants and one KA1 domain variant, indicating a (partial) loss-of-function effect. Interestingly, overexpressing BRSK2 with the remaining missense variant from the kinase domain and the two most C-terminal missense variants resulted in possible gain of function. Our findings further delineate the clinical and molecular spectrum of BRSK2 -associated NDD and provide further insights into the role of BRSK2/sff in nervous system function and dysfunction.

KBG syndrome (KBGS, OMIM #148050) is a rare genetic disorder caused by heterozygous truncating or missense variants in the ANKRD11 gene or a deletion of 16q24.3 involving ANKRD11 . While truncating variants clearly disrupt protein function, the interpretation of missense variants is more challenging, as many remain variants of uncertain significance (VUS). To address this, we evaluated PhenoScore, an open‐source AI‐based phenomics framework integrating facial recognition and medical data analysis, for predicting the pathogenicity of ANKRD11 missense variants and providing supporting evidence for variant interpretation within the ACMG framework, specifically the PP4 criterion. PhenoScore was trained on 79 individuals with truncating variants in ANKRD11 and age‐, sex‐, and ethnicity‐matched controls with other neurodevelopmental disorders, and its performance was compared to AlphaMissense, REVEL, and the evaluation of a clinical geneticist. Six individuals with functionally confirmed pathogenic missense variants were used for testing. PhenoScore achieved high predictive accuracy with an area under the curve (AUC) of 0.95 and a Brier score of 0.089, and pathogenic missense variants in the test set received a mean prediction score of 0.94. PhenoScore significantly outperformed REVEL ( p < 0.01), especially in cases supported by functional and clinical evidence, while no significant difference was observed compared to AlphaMissense ( p = 0.63); importantly, the two tools showed complementary strengths. These findings suggest that PhenoScore represents a promising tool for clinical variant interpretation, as it quantifies phenotypic concordance with KBGS and provides objective evidence that can strengthen the PP4 criterion within the ACMG framework. Combined with molecular prediction tools like AlphaMissense, PhenoScore may help reduce uncertainty surrounding VUS in ANKRD11 by complementing these scores.

Introduction: Birk-Barel syndrome (BIBARS), also known as KCNK9 imprinting syndrome, is an extremely rare genetic condition caused by pathogenic variants in the KCNK9 gene located on the long arm of chromosome 8 (8q24). This gene is paternally imprinted and therefore only expressed from the maternal allele. KCNK9 encodes for a member of the two-pore domain potassium channel (K2P) subfamily. BIBARS syndrome is characterized by congenital hypotonia and weakness of proximal muscles, intellectual disability, behavioural problems, dysmorphic features, feeding problems and epilepsy. The syndrome is implied in neurodevelopment, plays a role in various regulatory systems (eg, blood pressure maintenance, respiratory function, sleep, and cognitive function), and is associated with absence epilepsy. Methods: An institutionalized adult female patient with intellectual disability and challenging behaviours was presented whose history was carefully described and in whom extensive somatic, neurological, psychiatric and psychological investigation was performed, in addition to whole exome sequencing. Results: Intellectual disability, developmental delay with severe psychotrauma from early age on, as well as severe aggressive and self-injurious behaviours were established. Whole exome sequencing finally disclosed a pathogenic variant in the KCNK9 gene and an absence epilepsy in association with BIBARS was suspected. After treatment with valproic acid epileptic phenomena no longer occurred and behaviour and emotion regulation improved significantly. Discussion: A pathogenic heterozygous missense variant in the KCNK9 gene corresponding with a diagnosis of Birk-Barel syndrome was considered to be largely responsible for the severely disinhibited behaviours and causative for the absence epilepsy. For adequate diagnosis and treatment of neurodevelopmental disorders, this case clearly demonstrates the importance of adherence to clinical standards, in particular periodically renewed genetic analysis by means of WES/WGS.

KBG syndrome is a rare autosomal dominant neurodevelopmental disorder caused by ANKRD11 haploinsufficiency and is characterized by short stature, distinctive facial features, intellectual disability or developmental delay, congenital anomalies and skeletal anomalies. Although skeletal anomalies are reported in about 75% of cases, their nature and extent in relation to growth are largely unknown. Therefore, this study aims to asses the prevalence of skeletal anomalies in KBG syndrome and explore whether there is a relationship with short stature. This retrospective cohort study includes patients with a confirmed diagnosis of KBG syndrome, with available radiographic images at the Radboud University Medical Center. The radiographs were re‐evaluated by a radiologist focusing on the presence of spinal, costal, vertebral, and hand anomalies using standardized radiological criteria. In our cohort of 38 persons with KBG syndrome, 92% show skeletal anomalies on radiographic imaging. The most frequent observations on the radiographs were spinal anomalies (74%) and costal anomalies (40%). Specifically, lordosis (36%) had the highest prevalence. Patients with short stature ( n = 14) showed higher prevalences of kyphosis and delayed skeletal age. In conclusion, this study demonstrates a high prevalence and broad variety of skeletal anomalies in individuals with KBG syndrome.

Children with monogenic neurodevelopmental disorders often grow abnormally. Gene-specific growth charts would be useful but require large samples to construct them using the conventional LMS method. We transformed anthropometry to British 1990 reference z-scores for 328 UK and 264 international individuals with ANKRD11, ARID1B, ASXL3, DDX3X, KMT2A , or SATB2- related disorders, and modelled mean and standard deviation (SD) of the z-scores as gene-specific linear age trends adjusted for sex. Assuming the same skewness in the reference and rare disease distributions, we then back-transformed the mean ±2 SD lines to give gene-specific median, 2nd, and 98th centiles. The resulting z-score charts look plausible on several counts. Only KMT2A shows a (rising) age trend in median height, while BMI and weight increase for several genes, possibly reflecting population trends. Apart from SATB2 and DDX3X , the gene-specific medians are all below the reference (range 0.1th centile for height KMT2A to 36th centile for BMI ANKRD11 ). Median OFC z-score shows no age trend, with medians ranging from 10th to 30th centile, and ASXL3 is lowest, on the 3rd centile. In 19/24 cases, the centiles for the two sexes are the same on the z-score scale. Our LMSz method produces gene-specific growth charts for rare diseases, which, when used in the correct context, could be an important clinical tool. We plan to automate it within the DECIPHER platform, enabling availability for relevant genes.

KBG syndrome is one of the most frequent neurodevelopmental disorders and is caused by ANKRD11 variants. Postnatal short stature is observed in ~50% of patients. Recombinant human growth hormone (rhGH) has become a valuable treatment for patients with growth hormone deficiency (GHD) along with Prader–Willi and Turner syndrome. Limited evidence suggests benefits for some patients with KBG syndrome, but systematic analysis and detailed phenotyping are lacking. In this study, we include six unpublished patients with KBG syndrome treated with rhGH and 22 patients from the literature. At treatment start, 43% had GHD. Median height before treatment was −2.9 standard deviation score (SDS) which increased to −1.8 SDS at the latest measurement. Moreover, half of the patients showed height gains of ≥ 1 SDS and 10/18 patients with short stature before treatment achieved heights within normal ranges following treatment or at the latest available measurement. Of interest, the difference between pre‐treatment height SDS and that at the latest measurement (∆HSDS) was comparable for patients with and without GHD. This study represents the largest group of patients with KBG syndrome treated with rhGH so far and suggests that rhGH treatment has a positive impact on height, as indicated by ∆HSDS, in a subset of patients.

Natural History Studies can help inform clinician and caregiver expectations, form the basis of management guidelines, and provide a comparator for therapeutic intervention. In rare conditions, where collection of prospective longitudinal data is untimely and impractical, quasi‐natural history data—from multiple individuals of different ages—provides an alternative approach. A detailed genotype–phenotype analysis of 64 individuals with pathogenic or likely pathogenic ASXL3 variants was carried out, comprising qualitative and quantitative data. The majority of data was collected through direct clinic consultation with the individual and/−or caregiver(s). We report significant phenotypic variability, but improvement trends in feeding, hypotonia, verbalisation, and motor skills over time. Findings include: an increased prevalence of antenatal and neonatal structural anomalies, an emerging renal phenotype, a tendency for poor post‐natal growth (with novel reports of obesity later in childhood), and a lower‐than‐expected prevalence of seizures (compared to the existing literature). We also provide the first qualitative descriptions of several mildly affected probands, at different ages. Our recommendations include: baseline renal imaging after diagnosis, and Dental and Ophthalmological follow‐up for all. We describe the largest‐to‐date cohort of individuals with ASXL3 ‐related disorder, including 24 novel variants, novel clinical findings, quasi‐natural history trends, management insights, and recommendations.

DDX3X-related neurodevelopmental disorder is one of the most common monogenic causes of intellectual disability in females, with currently >1000 females diagnosed worldwide. In contrast, reports on affected males with DDX3X variants are scarce. The limited knowledge on this X-linked disorder in males hinders the interpretation of hemizygous DDX3X variants in clinical practice. In this study, we present a new cohort of 19 affected males (from 17 unrelated families) with (possibly) disease-causing DDX3X variants, for whom we collected clinical and molecular data. Additionally, we reviewed the existing literature on 13 males with DDX3X variants. The phenotype in males is diverse, including intellectual disability, speech/language delays, behavioural challenges and structural brain abnormalities. The vast majority of males have missense variants, including two recurrent variants (p.(Arg351Gln) and p.(Arg488Cys)). No truncating variants have been reported, consistent with the presumed embryonic lethality of complete loss-of-function of DDX3X in males. In our novel cohort, 6/17 variants are de novo in the affected male and 3/17 variants are de novo in the mother. This study provides significant insights in the genetic and phenotypic spectrum of males with DDX3X variants, by presenting the data of a combined cohort (n = 32) of novel and published individuals. Our data show that variants in DDX3X can cause an X-linked neurodevelopmental disorder in males, with unaffected or mildly affected carrier females. These findings will aid the interpretation of hemizygous missense variants in DDX3X and can guide clinical management and counselling, in particular with regard to recurrence risks in the respective families.

While mostly de novo truncating variants in SCAF4 were recently identified in 18 individuals with variable neurodevelopmental phenotypes, knowledge on the molecular and clinical spectrum is still limited. We assembled data on 50 novel individuals with SCAF4 variants ascertained via GeneMatcher and personal communication. With detailed evaluation of clinical data, in silico predictions and structural modeling, we further characterized the molecular and clinical spectrum of the autosomal dominant SCAF4-associated neurodevelopmental disorder. The molecular spectrum comprises 25 truncating, eight splice-site and five missense variants. While all other truncating variants were classified as pathogenic/likely pathogenic, significance of one C-terminal truncating variant, one splice-site variant and the missense variants remained unclear. Three missense variants in the CTD-interacting domain of SCAF4 were predicted to destabilize the domain. Twenty-three variants occurred de novo, and variants were inherited in 13 cases. Frequent clinical findings were mild developmental delay with speech impairment, seizures, and skeletal abnormalities such as clubfoot, scoliosis or hip dysplasia. Cognitive abilities ranged from normal IQ to severe intellectual disability (ID), with borderline to mild ID in the majority of individuals. Our study confirms the role of SCAF4 variants in neurodevelopmental disorders and further delineates the associated clinical phenotype.