PURPOSE:TCF7L2 (OMIM 602228; HGNC:11641) is a transcription factor and a critical effector of the Wnt/ β-Catenin pathway. In 2021, 11 pediatric patients with monoallelic predicted loss-of-function (pLOF) TCF7L2 variants and syndromic features were observed. Characterization of patients with pLOF TCF7L2 variants and neurodevelopmental features-herein referred to as TCF7L2-related neurodevelopmental disorder-is urgently needed. METHODS:We leveraged multiple methods (eg, GeneMatcher, DECIPHER, literature review, and public/private repositories) to identify an international cohort of 76 patients with pLOF TCF7L2 variants and neurodevelopmental features and phenotypically characterized them. We also retrospectively searched for an independent cohort of adults with pLOF TCF7L2 variants (n = 11) from more than 60,000 PennMedicine BioBank patients. RESULTS:Among 76 patients with pLOF TCF7L2 variants, speech delay (95.3%), craniofacial dysmorphisms (73.3%), ophthalmologic conditions (65.5%), autism (62.1%), and orthopedic abnormalities (52.6%) were the most commonly observed. Phenotypic differences did not cluster by variant type or genomic locus. Among PennMedicine BioBank patients, an association of nominal significance with type 2 diabetes with renal manifestations (odds ratio = 5.8; P = .03) was detected, warranting further investigation. CONCLUSION:This study represents the most comprehensive characterization of TCF7L2-related neurodevelopmental disorder to date, a novel neurodevelopmental disorder, defining its genotypic and phenotypic spectra. We opened a Simons Searchlight natural history study that is now available for patient enrollment to enhance the understanding of this condition.

Congenital myopathy-14 (CMYO14) is an ultrarare autosomal recessive disorder caused by biallelic variants in MYL1, with only four patients reported to date. We describe what is likely the fifth reported patient, a neonate with severe hypotonia, respiratory insufficiency, and skeletal anomalies showing distinct histological changes of skeletal muscle consistent with all previously described patients. The patient carried a novel homozygous intron variant (c.479-25T>C, p.(?)) in MYL1. RNA analysis of patient muscle demonstrated aberrant splicing, with inclusion of 19 bp from intron 4 in most transcripts, resulting in a frameshift and premature stop codon, and in-frame skipping of exons 4-5 in a minority of transcripts encompassing functional domains. In addition to core CMYO14 features, the patient presented with craniofacial anomalies not previously described. This case broadens the genotypic and phenotypic spectrum of MYL1-related disease and underscores the diagnostic importance of intron variants, highlighting the value of combining in silico splice prediction with functional RNA analyses.

ABSTRACT MRPS34 encodes a mitoribosomal protein essential for mitochondrial translation. Biallelic pathogenic variants in MRPS34 cause Combined Oxidative Phosphorylation Deficiency 32 (COXPD32), a rare mitochondrial disorder within the Leigh syndrome spectrum (LSS), ranging from fatal in infancy to adult survival. The objective is to describe two new individuals with MRPS34 ‐related disease and expand the clinical, genetic, and phenotypic spectrum of COXPD32. Clinical, radiological, biochemical, and molecular evaluations were conducted in two individuals with Leigh Syndrome (LS). Exome and genome sequencing identified presumed biallelic MRPS34 variants. A systematic review of all previously reported cases was performed to assess possible genotype–phenotype correlations ( n = 11). Individual 1, who died in infancy with LS, was presumed compound heterozygous for a novel splice‐site variant (c.364 + 2 T>C, p.(?)) and a nonsense variant (c.94C>T, p.(Gln32*)). Individual 2 survived into mid childhood and was homozygous for the hypomorphic variant c.322‐10G>A, p.(?). Among 11 individuals, key features included developmental delay (100%), lactic acidosis (91%), brainstem lesions (91%), and metabolic acidosis (83%). Homozygosity for c.322‐10G>A, p.(?) correlated with longer survival. MRPS34 ‐related disease presents with multisystemic features and genotype‐dependent severity. Accurate genetic diagnosis is essential for prognosis and therapeutic strategies.

AIM:To give a comprehensive overview of genetic findings in children with cerebral palsy, including a description of subtype, comorbidities and neuroimaging, providing insight into the clinical utility of genetics. METHOD:A systematic review of previous literature using Embase and Medline as databases. All studies were published between 2000 and 2022, each including at least 10 individuals with cerebral palsy. RESULTS:A total of 19 studies were included, comprising a total of 3707 individuals with cerebral palsy. The overall diagnostic yield was 22.2%, with the highest yield (up to 55%) in cryptogenic cerebral palsy. Variants in a total of 377 unique genes were identified, most frequently CTNNB1, SPAST and ATL1. In addition, 59 different CNVs were identified, of which 32 were in known (micro)duplication/-deletion syndromes such as 22q11.2 microdeletion/-duplication and 14q12 microdeletion. Spastic and dyskinetic cerebral palsy were the most common phenotypes among genetically diagnosed cases. Findings also included variants in genes linked to epilepsy, specific neuroimaging patterns and potentially treatable conditions. CONCLUSION:These results highlight the importance of genetic evaluation for diagnostic clarification, targeted treatment, monitoring of comorbidities and genetic counseling. We recommend offering genetic testing to individuals with cryptogenic cerebral palsy to optimize management and prevention.

Respiratory syncytial virus (RSV) is a major health problem worldwide, particularly in infants and young children. The infection can progress to life-threatening lower respiratory disease and, in rare cases, involves the central nervous system. We explore the pathophysiology in a child with high fever, seizures, and encephalopathy with brain inflammation during severe RSV infection. Whole-genome sequencing revealed homozygosity for a rare loss-of-function variant in the early-onset Parkinson-related gene PARK7/DJ-1. PARK7 plays a role in immune regulation, stress responses, and cell death. The patient’s Peripheral blood mononuclear cells and fibroblasts exhibited increased inflammatory cytokine responses, impaired RSV-induced apoptosis, and dampened autophagy. Studies in PARK7-deficient neuronal cells recapitulated the patient’s cellular phenotype, which was reversed upon PARK7 reconstitution. To our knowledge, this is the first association between PARK7 deficiency and RSV-induced brain inflammation, encephalopathy, and seizures. Collectively, our results demonstrate a role for PARK7 in regulation of inflammation and cellular homeostasis and suggest that PARK7 deficiency may aggravate infectious disease and cause immunopathology.

DDX3X dysfunction causes an X-linked multisystem disorder with high penetrance and variable expressivity. The phenotypic spectrum spans from learning disability without somatic involvement to profound intellectual disability with severe impairments in the central nervous system and other organs. A few multicenter studies and single case reports have previously highlighted some common phenotypic patterns but were unable to delineate correlations between underlying DDX3X variants and phenotypic findings.From the second largest patient cohort published to date, we analysed clinical, psychometric and diagnostic findings of 52 female and 7 male individuals, harbouring de novo and inherited DDX3X variants. These female patients revealed previously unknown quantitative correlations between variant type and localization and specific phenotypic findings (growth features, epilepsy, brain anomalies, dysmorphisms, motor-focused neurological findings). Moreover, by analysing the in silico folding and RNA binding capability of mutant DDX3X monomers, we were able to delineate novel correlations between DDX3X monomer misfolding grade and phenotypic severity.

INTRODUCTION:Untargeted metabolomics is a powerful tool for detecting perturbations in biological systems, offering significant potential for screening for rare inherited metabolic disorders (IMDs). However, the rarity and vast diversity of these diseases, results in limited availability of samples and incomplete metabolic pathway knowledge for each condition. Current diagnostic procedures rely heavily on manual interpretation, which is time-consuming, and data driven approaches are insufficient for small sample sizes. OBJECTIVES:To develop a diagnostic algorithm for IMDs addressing the challenges posed by small sample sizes and continuously evolving datasets. METHODS:77 IMD patients (35 different IMDs) and 136 control samples were collected from Copenhagen University Hospital, Rigshospitalet. The metabolome was analyzed using liquid chromatography-mass spectrometry. An algorithm partially based on sparse hierarchical clustering was designed to generate IMD-specific metabolic signatures from metabolomics data, enabling comparison with undiagnosed patient samples to provide diagnostic predictions. An iterative improvement strategy was employed, where new data are continuously integrated to refine the IMD-specific signatures. The algorithm's performance was evaluated through both the current study and a case study using literature-derived data. RESULTS:The algorithm demonstrated iterative improvement with each training round, correctly identifying the diagnosis within top 3 potential IMDs in 60% of samples (top 1 in 42%). The case study applied the method to literature-based data comprising 95 IMD samples (11 different IMDs) and 68 controls, yielding a correct diagnosis in 73.5% of cases. CONCLUSION:These results demonstrate that the algorithm provides a flexible, data-driven framework for continuous improvement in IMD diagnosis, even with limited number of samples.

Genetic testing plays a significant role in rare disease diagnostics. The most widespread technology for genetic testing of patients is next generation sequencing or second-generation sequencing, including whole exome sequencing (WES). Our laboratory performed diagnostic WES on 1660 samples representing 825 index patients aged 0-84 years between 2014 and 2020. The cohort is comprised of consecutive patients with a rare disease referred for diagnostic WES with analysis of all known disease genes. The main referrals were paediatric, clinical genetic and adult neurology departments. Patients' symptoms were translated to terms in the Human Phenotype Ontology (HPO) system and each symptom assigned to a single top-level HPO term. Variants were classified according to ACMG-AMP guidelines. The diagnostic yield in this cohort was 33.7 % with 278 patients receiving a genetic diagnosis. Patients with complex phenotypes with involvement of several organ systems were more likely to receive a genetic diagnosis. Higher diagnostic yields were seen for phenotypes including growth abnormalities, abnormalities of the ear or of the musculoskeletal system as well.

Nonsense-mediated mRNA decay (NMD) plays a crucial role in degrading aberrant transcripts with premature termination codons, with the Up-frameshift (UPF) protein family-UPF1, UPF2, and UPF3A/UPF3B-being vital components of this machinery. While several variants in genes encoding UPF2 and UPF3A/3B have been associated with neurodevelopmental disorders, only three germline UPF1 variants have been reported to date. Here, we report a male patient with a de novo missense variant, p.(Ala526Thr), in a highly conserved helicase motif of UPF1. The patient presented with moderate intellectual disability (ID), atypical autism, attention deficit hyperactivity disorder (ADHD), and behavioral disturbances. The common features observed among the four patients with UPF1 variants are moderate to severe ID and developmental delays in motor and verbal skills. A comparison across the disorders related to the UPF genes suggests that neurodevelopmental delay, including ID and impaired verbal skills, is a common feature, and these disorders may collectively be referred to as UPF-related neurodevelopmental disorders (NDDs). ADHD, autism, seizures, hypotonia, and non-specific dysmorphic features are also reported in some patients, suggesting that these disorders can be classified as non-specific intellectual disability syndromes. However, further studies are necessary to elucidate genotype-phenotype correlations and the molecular mechanisms underlying these rare disorders, particularly those related to UPF1.

Liver involvement in POLG disease is common and associated with high morbidity and mortality. Detailed, large-scale, systematic studies of liver involvement are lacking. This study aims to describe the onset, clinical course and prognostic implications of liver involvement in POLG disease. We conducted a multinational, retrospective study including clinical, genetic and biochemical data from patients with confirmed POLG disease. Patients were stratified according to age of disease onset: early-onset (< 12 years), juvenile/adult-onset (12-40 years), and late-onset (> 40 years). Of the 202 patients, 110 (54%) had liver involvement. This could present at any time during the lifespan, but occurred more frequently in patients with early-onset disease (76/98, 78%). Median onset age for liver involvement in females was 7 years (range: 1 month to 50 years), and 21 months in males (birth to 71 years). Infection-triggered disease onset carried a significantly higher risk of liver involvement than spontaneous or other disease triggers. Eighty-five percent of those with liver involvement also had epilepsy. Liver involvement was an indicator of poor prognosis and was significantly associated with worse survival. This study provides a comprehensive description of liver involvement in a large cohort of POLG disease patients. Liver involvement is common in this disease and associated with significantly worse survival. POLG disease should be considered in children presenting with liver involvement, and rapid genetic testing may guide management decisions. Our findings emphasize the need for early vigilance in monitoring liver involvement in all patients with confirmed POLG disease, particularly those with early-onset disease and during intercurrent infection.

Background and aims: Primary Coenzyme Q (CoQ) deficiency caused by COQ4 defects is a clinically heterogeneous mitochondrial condition characterized by reduced levels of CoQ10 in tissues. Next-generation sequencing has lately boosted the genetic diagnosis of an increasing number of patients. Still, functional validation of new variants of uncertain significance is essential for an adequate diagnosis, proper clinical management, treatment, and genetic counseling. Materials and methods: Both fibroblasts from a proband with COQ4 deficiency and a COQ4 knockout cell model have been characterized by a combination of biochemical and genetic analysis (HPLC lipid analysis, Oxygen consumption, minigene analysis, RNAseq, among others). Results: Here, we report the case of a subject harboring a new variant of the COQ4 gene in compound heterozygosis, which shows severe clinical manifestations. We present the molecular characterization of this new pathogenic variant affecting the splicing of COQ4. Conclusion: Our results highlight the importance of expanding the genetic analysis beyond the coding sequence to reduce the misdiagnosis of primary CoQ deficiency patients.

PURPOSE:Spastic paraplegia, intellectual disability, nystagmus, and obesity syndrome (SINO) is a rare autosomal dominant condition caused by heterozygous variants in KIDINS220. A total of 12 individuals are reported, comprising 8 with SINO and 4 with an autosomal recessive condition attributed to biallelic KIDINS220 variants. METHODS:In our international cohort, we have included 14 individuals, carrying 13 novel pathogenic KIDINS220 variants in heterozygous form. We assessed the clinical and molecular data of our cohort and previously reported individuals and, based on functional experiments, reached a better understanding of the pathogenesis behind the KIDINS220-related disease. RESULTS:Using fetal tissue and in vitro assays, we demonstrate that the variants generate KIDINS220 truncated forms that mislocalize in punctate intracellular structures, with decreased levels of the full-length protein, suggesting a trans-dominant negative effect. A total of 92% had their diagnosis within 3 years, with symptoms of developmental delay, spasticity, hypotonia, lack of eye contact, and nystagmus. We identified a KIDINS220 variant associated with fetal hydrocephalus and show that 58% of examined individuals present brain ventricular dilatation. We extend the phenotypic spectrum of SINO syndrome to behavioral manifestations not previously highlighted. CONCLUSION:Our study provides further insights into the clinical spectrum, etiology, and predicted functional impact of KIDINS220 variants.

Craniosynostosis constitutes one of the most common congenital cranial malformations, affecting approximately 6/10,0000 live births. A genetic etiology has long been known for several forms of syndromic craniosynostosis, including pathogenic variants in TWIST1 and FGFR3 in children with Saethre-Chotzen and Muenke syndrome. Over the last decade, reports of genetic aberrations in TCF12 in children with craniosynostosis have emerged, in particular in cases with premature closure of the coronal suture(s). In this study, we, therefore, systematically reviewed the rapidly growing knowledge of TCF12-related coronal craniosynostosis, clearly illustrating its high degree of genotype and phenotype variability. With the two novel cases presented, at least 113 cases of TCF12-related coronal craniosynostosis have currently been reported. By pooling data from several prospectively collected undifferentiated craniosynostosis cohorts (ntotal = 770), we estimate a prevalence of pathogenic TCF12 variants of at least 2

Purpose The aim of this study was to provide granular descriptions of aetiologies of hydrocephalus and concurrent conditions. Furthermore, to describe the short-term effect of ventriculoperitoneal shunt (VPS) and endoscopic third ventriculostomy (ETV) on preoperative symptoms and to estimate the risks of shunt revision, conversion of ETV to shunt, and the risks of complications. Methods A retrospective, single-centre study was performed among children who underwent shunt insertion or ETV for hydrocephalus between December 1st, 2016 and December 31st, 2021. Information regarding course of disease, surgical treatments, and comorbidities were registered from the digital patient records. The risks of conversion of treatment modality and shunt revision were estimated as cumulative incidences (CI’s) after 30-days, 3-, and 12 months. Results A total of 89 children with either obstructive (OH) (65%) or communicating (CH) (35%) hydrocephalus were surgically treated with VPS or ETV. Neurological disorders (CH 29%, OH 26%) and genetic conditions (CH 36%, OH 16%) were frequent concurrent conditions. Increased head size (74%) and symptoms of elevated intracranial pressure (57%) were the most common preoperative symptoms. Treatment with VPS entailed a 13.7% failure rate for OH and 9.7% for CH, whereas ETV displayed 14.3% failure rate, all reflecting failure of alleviating the preoperative clinical symptoms. The cumulative 1-year risk of shunt revision was 67%, most often due to valve dysfunction (28%), disconnection (14%), and shunt displacements (12%). Conclusion The considerable risks of treatment failure and complications call for improvements in treatment based on a better understanding of the heterogenic mechanisms of disease.

We aimed to provide a detailed phenotypic description of status epilepticus (SE) in a large cohort of patients with POLG disease and identify prognostic biomarkers to improve the management of this life-threatening condition. In a multinational, retrospective study with data on patients with POLG disease from seven European countries, we identified those who had SE. The age of SE onset, accompanying clinical, laboratory, imaging and genetic findings were analysed. One hundred and ninety-five patients with genetically confirmed POLG disease were recruited, of whom 67% (130/194) had epilepsy. SE was identified in 77% (97/126), with a median age of SE onset of 7 years. SE was the presenting symptom of the disease in 43% (40/93) of those with SE, while 57% (53/93) developed SE during the disease course. Convulsive SE was reported in 97% (91/94) followed by epilepsia partialis continua in 67% (56/84). Liver impairment 78% (74/95), ataxia 69% (60/87), stroke-like episodes 57% (50/88), were the major comorbidities. In the majority (66%; 57/86) with SE this became refractory or super-refractory. The presence of seizures was associated with significantly higher mortality compared to those without (P ≤ 0.001). The median time from SE debut to death was 5 months. SE is a major clinical feature of POLG disease in early and juvenile to adult-onset disease and can be the presenting feature or arise as part of a multisystem disease. It is associated with high morbidity and mortality, with the majority of patients with SE going on to develop refractory or super-refractory SE.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Objective Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. Methods We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. Results We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. Conclusions Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.

Alu elements are short, interspersed elements located throughout the genome, playing a role in human diversity, and occasionally causing genetic diseases. Here, we report a novel Alu insertion causing Mowat-Wilson syndrome, a rare neurodevelopmental disorder, in an 8-year-old boy displaying the typical clinical features for Mowat-Wilson syndrome. The variant was not initially detected in genome sequencing data, but through deep phenotyping, which pointed to only one plausible candidate gene, manual inspection of genome sequencing alignment data enabled us to identify a de novo heterozygous Alu insertion in exon 8 of the ZEB2 gene. Nanopore long-read sequencing confirmed the Alu insertion, leading to the formation of a premature stop codon and likely haploinsufficiency of ZEB2. This underscores the importance of deep phenotyping and mobile element insertion analysis in uncovering genetic causes of monogenic disorders as these elements might be overlooked in standard next-generation sequencing protocols.