Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes

Genotype; Intellectual disability; PhenotypeGenotipo; Discapacidad intelectual; FenotipoGenotip; Discapacitat intel·lectual; FenotipWe describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz–Kaveggia syndrome, Lujan–Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype–phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations.Foundation for Science and Technology (FCT), Grant/Award Number: UIDB/00215/2020 UIDP/00215/2020 LA/P/0064/2020; Broad Institute; National Eye Institute; National Heart, Lung and Blood Institute, Grant/Award Number: UM1 HG008900; National Human Genome Research Institute, Grant/Award Number: R01 HG009141

Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1-Related Disorder Patient Neurons

Background: STXBP1-related disorder (STXBP1-RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability, and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of neurons of patients with STXBP1-RD are unknown. Methods: We combined live-cell imaging, electrophysiology, confocal microscopy, and mass spectrometry proteomics to characterize cellular phenotypes of induced pluripotent stem cell–derived neurons from 6 patients with STXBP1-RD, capturing shared features as well as phenotypic diversity among patients. Results: Neurons from all patients showed normal in vitro development, morphology, and synapse formation, but reduced MUNC18-1 RNA and protein levels. In addition, a proteome-wide screen identified dysregulation of proteins related to synapse function and RNA processes. Neuronal networks showed shared as well as patient-specific phenotypes in activity frequency, network irregularity, and synchronicity, especially when networks were challenged by increasing excitability. No shared effects were observed in synapse physiology of single neurons except for a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested 2 patient clusters, not explained by gene variant type. Conclusions: Together, these data show that decreased MUNC18-1 levels, dysregulation of synaptic proteins, and altered network activity are shared cellular phenotypes of STXBP1-RD. The 2 patient clusters suggest distinctive pathobiology among subgroups of patients, providing a plausible explanation for the clinical heterogeneity. This phenotypic spectrum provides a framework for future validation studies and therapy design for STXBP1-RD

Reduced MUNC18-1 Levels, Synaptic Proteome Changes, and Altered Network Activity in STXBP1-Related Disorder Patient Neurons

Background: STXBP1-related disorder (STXBP1-RD) is a neurodevelopmental disorder caused by pathogenic variants in the STXBP1 gene. Its gene product MUNC18-1 organizes synaptic vesicle exocytosis and is essential for synaptic transmission. Patients present with developmental delay, intellectual disability, and/or epileptic seizures, with high clinical heterogeneity. To date, the cellular deficits of neurons of patients with STXBP1-RD are unknown. Methods: We combined live-cell imaging, electrophysiology, confocal microscopy, and mass spectrometry proteomics to characterize cellular phenotypes of induced pluripotent stem cell–derived neurons from 6 patients with STXBP1-RD, capturing shared features as well as phenotypic diversity among patients. Results: Neurons from all patients showed normal in vitro development, morphology, and synapse formation, but reduced MUNC18-1 RNA and protein levels. In addition, a proteome-wide screen identified dysregulation of proteins related to synapse function and RNA processes. Neuronal networks showed shared as well as patient-specific phenotypes in activity frequency, network irregularity, and synchronicity, especially when networks were challenged by increasing excitability. No shared effects were observed in synapse physiology of single neurons except for a few patient-specific phenotypes. Similarities between functional and proteome phenotypes suggested 2 patient clusters, not explained by gene variant type. Conclusions: Together, these data show that decreased MUNC18-1 levels, dysregulation of synaptic proteins, and altered network activity are shared cellular phenotypes of STXBP1-RD. The 2 patient clusters suggest distinctive pathobiology among subgroups of patients, providing a plausible explanation for the clinical heterogeneity. This phenotypic spectrum provides a framework for future validation studies and therapy design for STXBP1-RD.</p

STXBP1 Syndrome Is Characterized by Inhibition-Dominated Dynamics of Resting-State EEG

STXBP1 syndrome is a rare neurodevelopmental disorder caused by heterozygous variants in the STXBP1 gene and is characterized by psychomotor delay, early-onset developmental delay, and epileptic encephalopathy. Pathogenic STXBP1 variants are thought to alter excitation-inhibition (E/I) balance at the synaptic level, which could impact neuronal network dynamics; however, this has not been investigated yet. Here, we present the first EEG study of patients with STXBP1 syndrome to quantify the impact of the synaptic E/I dysregulation on ongoing brain activity. We used high-frequency-resolution analyses of classical and recently developed methods known to be sensitive to E/I balance. EEG was recorded during eyes-open rest in children with STXBP1 syndrome (n = 14) and age-matched typically developing children (n = 50). Brain-wide abnormalities were observed in each of the four resting-state measures assessed here: (i) slowing of activity and increased low-frequency power in the range 1.75-4.63 Hz, (ii) increased long-range temporal correlations in the 11-18 Hz range, (iii) a decrease of our recently introduced measure of functional E/I ratio in a similar frequency range (12-24 Hz), and (iv) a larger exponent of the 1/f-like aperiodic component of the power spectrum. Overall, these findings indicate that large-scale brain activity in STXBP1 syndrome exhibits inhibition-dominated dynamics, which may be compensatory to counteract local circuitry imbalances expected to shift E/I balance toward excitation, as observed in preclinical models. We argue that quantitative EEG investigations in STXBP1 and other neurodevelopmental disorders are a crucial step to understand large-scale functional consequences of synaptic E/I perturbations

DNA methylation episignature and comparative epigenomic profiling of HNRNPU-related neurodevelopmental disorder

Purpose: HNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder. Methods: DNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures. Results: A robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders. Conclusion: This study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test

DNA methylation episignature and comparative epigenomic profiling of HNRNPU-related neurodevelopmental disorder

Purpose: HNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder. Methods: DNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures. Results: A robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders. Conclusion: This study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test

Missense MED12 variants in 22 males with intellectual disability: From nonspecific symptoms to complete syndromes

We describe the phenotype of 22 male patients (20 probands) carrying a hemizygous missense variant in MED12. The phenotypic spectrum is very broad ranging from nonspecific intellectual disability (ID) to the three well-known syndromes: Opitz–Kaveggia syndrome, Lujan–Fryns syndrome, or Ohdo syndrome. The identified variants were randomly distributed throughout the gene (p = 0.993, χ2 test), but mostly outside the functional domains (p = 0.004; χ2 test). Statistical analyses did not show a correlation between the MED12-related phenotypes and the locations of the variants (p = 0.295; Pearson correlation), nor the protein domain involved (p = 0.422; Pearson correlation). In conclusion, establishing a genotype–phenotype correlation in MED12-related diseases remains challenging. Therefore, we think that patients with a causative MED12 variant are currently underdiagnosed due to the broad patients' clinical presentations

Episignature Mapping of TRIP12 Provides Functional Insight into Clark–Baraitser Syndrome

Clark–Baraitser syndrome is a rare autosomal dominant intellectual disability syndrome caused by pathogenic variants in the TRIP12 (Thyroid Hormone Receptor Interactor 12) gene. TRIP12 encodes an E3 ligase in the ubiquitin pathway. The ubiquitin pathway includes activating E1, conjugating E2 and ligating E3 enzymes which regulate the breakdown and sorting of proteins. This enzymatic pathway is crucial for physiological processes. A significant proportion of TRIP12 variants are currently classified as variants of unknown significance (VUS). Episignatures have been shown to represent a powerful diagnostic tool to resolve inconclusive genetic findings for Mendelian disorders and to re-classify VUSs. Here, we show the results of DNA methylation episignature analysis in 32 individuals with pathogenic, likely pathogenic and VUS variants in TRIP12. We identified a specific and sensitive DNA methylation (DNAm) episignature associated with pathogenic TRIP12 variants, establishing its utility as a clinical biomarker for Clark–Baraitser syndrome. In addition, we performed analysis of differentially methylated regions as well as functional correlation of the TRIP12 genome-wide methylation profile with the profiles of 56 additional neurodevelopmental disorders

Episignature Mapping of TRIP12 Provides Functional Insight into Clark&ndash;Baraitser Syndrome

Clark&ndash;Baraitser syndrome is a rare autosomal dominant intellectual disability syndrome caused by pathogenic variants in the TRIP12 (Thyroid Hormone Receptor Interactor 12) gene. TRIP12 encodes an E3 ligase in the ubiquitin pathway. The ubiquitin pathway includes activating E1, conjugating E2 and ligating E3 enzymes which regulate the breakdown and sorting of proteins. This enzymatic pathway is crucial for physiological processes. A significant proportion of TRIP12 variants are currently classified as variants of unknown significance (VUS). Episignatures have been shown to represent a powerful diagnostic tool to resolve inconclusive genetic findings for Mendelian disorders and to re-classify VUSs. Here, we show the results of DNA methylation episignature analysis in 32 individuals with pathogenic, likely pathogenic and VUS variants in TRIP12. We identified a specific and sensitive DNA methylation (DNAm) episignature associated with pathogenic TRIP12 variants, establishing its utility as a clinical biomarker for Clark&ndash;Baraitser syndrome. In addition, we performed analysis of differentially methylated regions as well as functional correlation of the TRIP12 genome-wide methylation profile with the profiles of 56 additional neurodevelopmental disorders

Assessing the landscape of STXBP1-related disorders in 534 individuals

Disease-causing variants in STXBP1 are among the most common genetic causes of neurodevelopmental disorders. However, the phenotypic spectrum in STXBP1-related disorders is wide and clear correlations between variant type and clinical features have not been observed so far. Here, we harmonized clinical data across 534 individuals with STXBP1-related disorders and analysed 19 973 derived phenotypic terms, including phenotypes of 253 individuals previously unreported in the scientific literature. The overall phenotypic landscape in STXBP1-related disorders is characterized by neurodevelopmental abnormalities in 95% and seizures in 89% of individuals, including focal-onset seizures as the most common seizure type (47%). More than 88% of individuals with STXBP1-related disorders have seizure onset in the first year of life, including neonatal seizure onset in 47%. Individuals with protein-truncating variants and deletions in STXBP1 (n = 261) were almost twice as likely to present with West syndrome and were more phenotypically similar than expected by chance. Five genetic hotspots with recurrent variants were identified in more than 10 individuals, including p.Arg406Cys/His (n = 40), p.Arg292Cys/His/Leu/Pro (n = 30), p.Arg551Cys/Gly/His/Leu (n = 24), p.Pro139Leu (n = 12), and p.Arg190Trp (n = 11). None of the recurrent variants were significantly associated with distinct electroclinical syndromes, single phenotypic features, or showed overall clinical similarity, indicating that the baseline variability in STXBP1-related disorders is too high for discrete phenotypic subgroups to emerge. We then reconstructed the seizure history in 62 individuals with STXBP1-related disorders in detail, retrospectively assigning seizure type and seizure frequency monthly across 4433 time intervals, and retrieved 251 anti-seizure medication prescriptions from the electronic medical records. We demonstrate a dynamic pattern of seizure control and complex interplay with response to specific medications particularly in the first year of life when seizures in STXBP1-related disorders are the most prominent. Adrenocorticotropic hormone and phenobarbital were more likely to initially reduce seizure frequency in infantile spasms and focal seizures compared to other treatment options, while the ketogenic diet was most effective in maintaining seizure freedom. In summary, we demonstrate how the multidimensional spectrum of phenotypic features in STXBP1-related disorders can be assessed using a computational phenotype framework to facilitate the development of future precision-medicine approaches