Pathogenic variants in KMT2C result in a neurodevelopmental disorder distinct from Kleefstra and Kabuki syndromes.

Trithorax-related H3K4 methyltransferases, KMT2C and KMT2D, are critical epigenetic modifiers. Haploinsufficiency of KMT2C was only recently recognized as a cause of neurodevelopmental disorder (NDD), so the clinical and molecular spectrums of the KMT2C-related NDD (now designated as Kleefstra syndrome 2) are largely unknown. We ascertained 98 individuals with rare KMT2C variants, including 75 with protein-truncating variants (PTVs). Notably, ∼15% of KMT2C PTVs were inherited. Although the most highly expressed KMT2C transcript consists of only the last four exons, pathogenic PTVs were found in almost all the exons of this large gene. KMT2C variant interpretation can be challenging due to segmental duplications and clonal hematopoesis-induced artifacts. Using samples from 27 affected individuals, divided into discovery and validation cohorts, we generated a moderate strength disorder-specific KMT2C DNA methylation (DNAm) signature and demonstrate its utility in classifying non-truncating variants. Based on 81 individuals with pathogenic/likely pathogenic variants, we demonstrate that the KMT2C-related NDD is characterized by developmental delay, intellectual disability, behavioral and psychiatric problems, hypotonia, seizures, short stature, and other comorbidities. The facial module of PhenoScore, applied to photographs of 34 affected individuals, reveals that the KMT2C-related facial gestalt is significantly different from the general NDD population. Finally, using PhenoScore and DNAm signatures, we demonstrate that the KMT2C-related NDD is clinically and epigenetically distinct from Kleefstra and Kabuki syndromes. Overall, we define the clinical features, molecular spectrum, and DNAm signature of the KMT2C-related NDD and demonstrate they are distinct from Kleefstra and Kabuki syndromes highlighting the need to rename this condition.

Epigenotype-genotype-phenotype correlations in SETD1A and SETD2 chromatin disorders.

Germline pathogenic variants in two genes encoding the lysine-specific histone methyltransferase genes SETD1A and SETD2 are associated with neurodevelopmental disorders (NDDs) characterized by developmental delay and congenital anomalies. The SETD1A and SETD2 gene products play a critical role in chromatin-mediated regulation of gene expression. Specific methylation episignatures have been detected for a range of chromatin gene-related NDDs and have impacted clinical practice by improving the interpretation of variant pathogenicity. To investigate if SETD1A and/or SETD2-related NDDs are associated with a detectable episignature, we undertook targeted genome-wide methylation profiling of > 2 M CpGs using a next-generation sequencing-based assay. A comparison of methylation profiles in patients with SETD1A variants (n = 6) did not reveal evidence of a strong methylation episignature. A review of the clinical and genetic features of the SETD2 patient group revealed that, as reported previously, there were phenotypic differences between patients with truncating mutations (n = 4, Luscan-Lumish syndrome; MIM:616831) and those with missense codon 1740 variants [p.Arg1740Trp (n = 4) and p.Arg1740Gln (n = 2)]. Both SETD2 subgroups demonstrated a methylation episignature, which was characterized by hypomethylation and hypermethylation events, respectively. Within the codon 1740 subgroup, both the methylation changes and clinical phenotype were more severe in those with p.Arg1740Trp variants. We also noted that two of 10 cases with a SETD2-NDD had developed a neoplasm. These findings reveal novel epigenotype-genotype-phenotype correlations in SETD2-NDDs and predict a gain-of-function mechanism for SETD2 codon 1740 pathogenic variants.

Integrative genetic analysis: cornerstone of precision psychiatry.

The role of genetic testing in the domain of neurodevelopmental and psychiatric disorders (NPDs) is gradually changing from providing etiological explanation for the presence of NPD phenotypes to also identifying young individuals at high risk of developing NPDs before their clinical manifestation. In clinical practice, the latter implies a shift towards the availability of individual genetic information predicting a certain liability to develop an NPD (e.g., autism, intellectual disability, psychosis etc.). The shift from mostly a posteriori explanation to increasingly a priori risk prediction is the by-product of the systematic implementation of whole exome or genome sequencing as part of routine diagnostic work-ups during the neonatal and prenatal periods. This rapid uptake of genetic testing early in development has far-reaching consequences for psychiatry: Whereas until recently individuals would come to medical attention because of signs of abnormal developmental and/or behavioral symptoms, increasingly, individuals are presented based on genetic liability for NPD outcomes before NPD symptoms emerge. This novel clinical scenario, while challenging, also creates opportunities for research on prevention interventions and precision medicine approaches. Here, we review why optimization of individual risk prediction is a key prerequisite for precision medicine in the sphere of NPDs, as well as the technological and statistical methods required to achieve this ambition.

Neurocognitive profiles of 22q11.2 and 16p11.2 deletions and duplications.

Rare recurrent copy number variants (CNVs) at chromosomal loci 22q11.2 and 16p11.2 are genetic disorders with lifespan risk for neuropsychiatric disorders. Microdeletions and duplications are associated with neurocognitive deficits, yet few studies compared these groups using the same measures to address confounding measurement differences. We report a prospective international collaboration applying the same computerized neurocognitive assessment, the Penn Computerized Neurocognitive Battery (CNB), administered in a multi-site study on rare genomic disorders: 22q11.2 deletions (n = 492); 22q11.2 duplications (n = 106); 16p11.2 deletion (n = 117); and 16p11.2 duplications (n = 46). Domains examined include executive functions, episodic memory, complex cognition, social cognition, and psychomotor speed. Accuracy and speed for each domain were included as dependent measures in a mixed-model repeated measures analysis. Locus (22q11.2, 16p11.2) and Copy number (deletion/duplication) were grouping factors and Measure (accuracy, speed) and neurocognitive domain were repeated measures factors, with Sex and Site as covariates. We also examined correlation with IQ. We found a significant Locus × Copy number × Domain × Measure interaction (p = 0.0004). 22q11.2 deletions were associated with greater performance accuracy deficits than 22q11.2 duplications, while 16p11.2 duplications were associated with greater specific deficits than 16p11.2 deletions. Duplications at both loci were associated with reduced speed compared to deletions. Performance profiles differed among the groups with particularly poor memory performance of the 22q11.2 deletion group while the 16p11.2 duplication group had greatest deficits in complex cognition. Average accuracy on the CNB was moderately correlated with Full Scale IQ. Deletions and duplications of 22q11.2 and 16p11.2 have differential effects on accuracy and speed of neurocognition indicating locus specificity of performance profiles. These profile differences can help inform mechanistic substrates to heterogeneity in presentation and outcome, and can only be established in large-scale international consortia using the same neurocognitive assessment. Future studies could aim to link performance profiles to clinical features and brain function.

Further delineation of the rare GDACCF (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies syndrome): genotype and phenotype of 22 patients with ZNF148 mutations.

BACKGROUND: Pathogenic variants in the zinc finger protein coding genes are rare causes of intellectual disability and congenital malformations. Mutations in the ZNF148 gene causing GDACCF syndrome (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies; MIM #617260) have been reported in five individuals so far. METHODS: As a result of an international collaboration using GeneMatcher Phenome Central Repository and personal communications, here we describe the clinical and molecular genetic characteristics of 22 previously unreported individuals. RESULTS: The core clinical phenotype is characterised by developmental delay particularly in the domain of speech development, postnatal growth retardation, microcephaly and facial dysmorphism. Corpus callosum abnormalities appear less frequently than suggested by previous observations. The identified mutations concerned nonsense or frameshift variants that were mainly located in the last exon of the ZNF148 gene. Heterozygous deletion including the entire ZNF148 gene was found in only one case. Most mutations occurred de novo, but were inherited from an affected parent in two families. CONCLUSION: The GDACCF syndrome is clinically diverse, and a genotype-first approach, that is, exome sequencing is recommended for establishing a genetic diagnosis rather than a phenotype-first approach. However, the syndrome may be suspected based on some recurrent, recognisable features. Corpus callosum anomalies were not as constant as previously suggested, we therefore recommend to replace the term 'GDACCF syndrome' with 'ZNF148-related neurodevelopmental disorder'.

Spatial control of nucleoporin condensation by fragile X-related proteins.

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.

The contribution of copy number variants to psychiatric symptoms and cognitive ability.

Copy number variants (CNVs) are deletions and duplications of DNA sequence. The most frequently studied CNVs, which are described in this review, are recurrent CNVs that occur in the same locations on the genome. These CNVs have been strongly implicated in neurodevelopmental disorders, namely autism spectrum disorder (ASD), intellectual disability (ID), and developmental delay (DD), but also in schizophrenia. More recent work has also shown that CNVs increase risk for other psychiatric disorders, namely, depression, bipolar disorder, and post-traumatic stress disorder. Many of the same CNVs are implicated across all of these disorders, and these neuropsychiatric CNVs are also associated with cognitive ability in the general population, as well as with structural and functional brain alterations. Neuropsychiatric CNVs also show incomplete penetrance, such that carriers do not always develop any psychiatric disorder, and may show only mild symptoms, if any. Variable expressivity, whereby the same CNVs are associated with many different phenotypes of varied severity, also points to highly complex mechanisms underlying disease risk in CNV carriers. Comprehensive and longitudinal phenotyping studies of individual CNVs have provided initial insights into these mechanisms. However, more work is needed to estimate and predict the effect of non-recurrent, ultra-rare CNVs, which also contribute to psychiatric and cognitive outcomes. Moreover, delineating the broader phenotypic landscape of neuropsychiatric CNVs in both clinical and general population cohorts may also offer important mechanistic insights.

Brain functional connectivity mirrors genetic pleiotropy in psychiatric conditions.

Pleiotropy occurs when a genetic variant influences more than one trait. This is a key property of the genomic architecture of psychiatric disorders and has been observed for rare and common genomic variants. It is reasonable to hypothesize that the microscale genetic overlap (pleiotropy) across psychiatric conditions and cognitive traits may lead to similar overlaps at the macroscale brain level such as large-scale brain functional networks. We took advantage of brain connectivity, measured by resting-state functional MRI to measure the effects of pleiotropy on large-scale brain networks, a putative step from genes to behaviour. We processed nine resting-state functional MRI datasets including 32 726 individuals and computed connectome-wide profiles of seven neuropsychiatric copy-number-variants, five polygenic scores, neuroticism and fluid intelligence as well as four idiopathic psychiatric conditions. Nine out of 19 pairs of conditions and traits showed significant functional connectivity correlations (rFunctional connectivity), which could be explained by previously published levels of genomic (rGenetic) and transcriptomic (rTranscriptomic) correlations with moderate to high concordance: rGenetic-rFunctional connectivity = 0.71 [0.40-0.87] and rTranscriptomic-rFunctional connectivity = 0.83 [0.52; 0.94]. Extending this analysis to functional connectivity profiles associated with rare and common genetic risk showed that 30 out of 136 pairs of connectivity profiles were correlated above chance. These similarities between genetic risks and psychiatric disorders at the connectivity level were mainly driven by the overconnectivity of the thalamus and the somatomotor networks. Our findings suggest a substantial genetic component for shared connectivity profiles across conditions and traits, opening avenues to delineate general mechanisms-amenable to intervention-across psychiatric conditions and genetic risks.

Insufficient Evidence for "Autism-Specific" Genes.

Despite evidence that deleterious variants in the same genes are implicated across multiple neurodevelopmental and neuropsychiatric disorders, there has been considerable interest in identifying genes that, when mutated, confer risk that is largely specific for autism spectrum disorder (ASD). Here, we review the findings and limitations of recent efforts to identify relatively "autism-specific" genes, efforts which focus on rare variants of large effect size that are thought to account for the observed phenotypes. We present a divergent interpretation of published evidence; discuss practical and theoretical issues related to studying the relationships between rare, large-effect deleterious variants and neurodevelopmental phenotypes; and describe potential future directions of this research. We argue that there is currently insufficient evidence to establish meaningful ASD specificity of any genes based on large-effect rare-variant data.

Rare copy number variation in posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is a heritable (h2 = 24-71%) psychiatric illness. Copy number variation (CNV) is a form of rare genetic variation that has been implicated in the etiology of psychiatric disorders, but no large-scale investigation of CNV in PTSD has been performed. We present an association study of CNV burden and PTSD symptoms in a sample of 114,383 participants (13,036 cases and 101,347 controls) of European ancestry. CNVs were called using two calling algorithms and intersected to a consensus set. Quality control was performed to remove strong outlier samples. CNVs were examined for association with PTSD within each cohort using linear or logistic regression analysis adjusted for population structure and CNV quality metrics, then inverse variance weighted meta-analyzed across cohorts. We examined the genome-wide total span of CNVs, enrichment of CNVs within specified gene-sets, and CNVs overlapping individual genes and implicated neurodevelopmental regions. The total distance covered by deletions crossing over known neurodevelopmental CNV regions was significant (beta = 0.029, SE = 0.005, P = 6.3 × 10-8). The genome-wide neurodevelopmental CNV burden identified explains 0.034% of the variation in PTSD symptoms. The 15q11.2 BP1-BP2 microdeletion region was significantly associated with PTSD (beta = 0.0206, SE = 0.0056, P = 0.0002). No individual significant genes interrupted by CNV were identified. 22 gene pathways related to the function of the nervous system and brain were significant in pathway analysis (FDR q < 0.05), but these associations were not significant once NDD regions were removed. A larger sample size, better detection methods, and annotated resources of CNV are needed to explore this relationship further.

Neurodevelopmental disorders (NDD) without boundaries: research and interventions beyond classifications.

On June 2022, the 2nd Webinar "Neurodevelopmental Disorders (NDD) without boundaries took place at the Imagine Institute in Paris and was broadcasted live and in replay. The aim of this webinar is to address NDD in a dimensional rather than in a categorical approach. Several speakers were invited to present their researches on the subject. Classifications in NDD were discussed: irritability in NDD, involvement of the immune system in neurodevelopment, nutrition and gut microbiota modulate brain inflammation and neurodevelopment, co-occurring conditions in autistic adolescents and adults without intellectual disability. Classifications in psychiatric disorders were asked: mapping the effect of genes on cognition and autism risk, epigenetics and symptomatic trajectory in neurodevelopmental disorders, the autism-schizophrenia continuum in two examples: minor neurological signs and EEG microstates, the cerebellum in schizophrenia and autism: from imaging to intervention perspectives. Both genetic and environmental factors, along with clinical and imaging features, argue toward a continnum between NDD but also with adult psychiatric presentations. This new paradigm could modify the therapeutic strategy, with the development of large-spectrum treatments or new psychotherapies addressing co-occuring symptoms. The complexity and the heterogeneity of NDD apply well to the next scientific and political challenges: developing international convergence to push back the frontiers of our knowledge. This article is a summary of the 2nd webinar "Neurodevelopmental Disorders (NDD) without boundaries: research and interventions beyond classifications" sponsored by the French National Academy of Medicine, the autism and neurodevelopmental disorders scientific interest group (GIS), the International Research Network Dev-O-Psy and the French Institute of Psychiatry (GDR3557). Oral presentations are available as a replay on the following website (in French): https://autisme-neurodev.org/evenements/2022/04/12/tnd-sans-frontieres-la-recherche-et-les-interventions-au-dela-des-classifications/ .

Conditional canonical correlation estimation based on covariates with random forests.

MOTIVATION: Investigating the relationships between two sets of variables helps to understand their interactions and can be done with canonical correlation analysis (CCA). However, the correlation between the two sets can sometimes depend on a third set of covariates, often subject-related ones such as age, gender or other clinical measures. In this case, applying CCA to the whole population is not optimal and methods to estimate conditional CCA, given the covariates, can be useful. RESULTS: We propose a new method called Random Forest with Canonical Correlation Analysis (RFCCA) to estimate the conditional canonical correlations between two sets of variables given subject-related covariates. The individual trees in the forest are built with a splitting rule specifically designed to partition the data to maximize the canonical correlation heterogeneity between child nodes. We also propose a significance test to detect the global effect of the covariates on the relationship between two sets of variables. The performance of the proposed method and the global significance test is evaluated through simulation studies that show it provides accurate canonical correlation estimations and well-controlled Type-1 error. We also show an application of the proposed method with EEG data. AVAILABILITY AND IMPLEMENTATION: RFCCA is implemented in a freely available R package on CRAN (https://CRAN.R-project.org/package=RFCCA). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide analysis of gene dosage in 24,092 individuals estimates that 10,000 genes modulate cognitive ability.

Genomic copy number variants (CNVs) are routinely identified and reported back to patients with neuropsychiatric disorders, but their quantitative effects on essential traits such as cognitive ability are poorly documented. We have recently shown that the effect size of deletions on cognitive ability can be statistically predicted using measures of intolerance to haploinsufficiency. However, the effect sizes of duplications remain unknown. It is also unknown if the effect of multigenic CNVs are driven by a few genes intolerant to haploinsufficiency or distributed across tolerant genes as well. Here, we identified all CNVs > 50 kilobases in 24,092 individuals from unselected and autism cohorts with assessments of general intelligence. Statistical models used measures of intolerance to haploinsufficiency of genes included in CNVs to predict their effect size on intelligence. Intolerant genes decrease general intelligence by 0.8 and 2.6 points of intelligence quotient when duplicated or deleted, respectively. Effect sizes showed no heterogeneity across cohorts. Validation analyses demonstrated that models could predict CNV effect sizes with 78% accuracy. Data on the inheritance of 27,766 CNVs showed that deletions and duplications with the same effect size on intelligence occur de novo at the same frequency. We estimated that around 10,000 intolerant and tolerant genes negatively affect intelligence when deleted, and less than 2% have large effect sizes. Genes encompassed in CNVs were not enriched in any GOterms but gene regulation and brain expression were GOterms overrepresented in the intolerant subgroup. Such pervasive effects on cognition may be related to emergent properties of the genome not restricted to a limited number of biological pathways.

Dissecting autism and schizophrenia through neuroimaging genomics.

Neuroimaging genomic studies of autism spectrum disorder and schizophrenia have mainly adopted a 'top-down' approach, beginning with the behavioural diagnosis, and moving down to intermediate brain phenotypes and underlying genetic factors. Advances in imaging and genomics have been successfully applied to increasingly large case-control studies. As opposed to diagnostic-first approaches, the bottom-up strategy begins at the level of molecular factors enabling the study of mechanisms related to biological risk, irrespective of diagnoses or clinical manifestations. The latter strategy has emerged from questions raised by top-down studies: why are mutations and brain phenotypes over-represented in individuals with a psychiatric diagnosis? Are they related to core symptoms of the disease or to comorbidities? Why are mutations and brain phenotypes associated with several psychiatric diagnoses? Do they impact a single dimension contributing to all diagnoses? In this review, we aimed at summarizing imaging genomic findings in autism and schizophrenia as well as neuropsychiatric variants associated with these conditions. Top-down studies of autism and schizophrenia identified patterns of neuroimaging alterations with small effect-sizes and an extreme polygenic architecture. Genomic variants and neuroimaging patterns are shared across diagnostic categories suggesting pleiotropic mechanisms at the molecular and brain network levels. Although the field is gaining traction; characterizing increasingly reproducible results, it is unlikely that top-down approaches alone will be able to disentangle mechanisms involved in autism or schizophrenia. In stark contrast with top-down approaches, bottom-up studies showed that the effect-sizes of high-risk neuropsychiatric mutations are equally large for neuroimaging and behavioural traits. Low specificity has been perplexing with studies showing that broad classes of genomic variants affect a similar range of behavioural and cognitive dimensions, which may be consistent with the highly polygenic architecture of psychiatric conditions. The surprisingly discordant effect sizes observed between genetic and diagnostic first approaches underscore the necessity to decompose the heterogeneity hindering case-control studies in idiopathic conditions. We propose a systematic investigation across a broad spectrum of neuropsychiatric variants to identify putative latent dimensions underlying idiopathic conditions. Gene expression data on temporal, spatial and cell type organization in the brain have also considerable potential for parsing the mechanisms contributing to these dimensions' phenotypes. While large neuroimaging genomic datasets are now available in unselected populations, there is an urgent need for data on individuals with a range of psychiatric symptoms and high-risk genomic variants. Such efforts together with more standardized methods will improve mechanistically informed predictive modelling for diagnosis and clinical outcomes.

Estimating the effects of copy-number variants on intelligence using hierarchical Bayesian models.

It is challenging to estimate the phenotypic impact of the structural genome changes known as copy-number variations (CNVs), since there are many unique CNVs which are nonrecurrent, and most are too rare to be studied individually. In recent work, we found that CNV-aggregated genomic annotations, that is, specifically the intolerance to mutation as measured by the pLI score (probability of being loss-of-function intolerant), can be strong predictors of intellectual quotient (IQ) loss. However, this aggregation method only estimates the individual CNV effects indirectly. Here, we propose the use of hierarchical Bayesian models to directly estimate individual effects of rare CNVs on measures of intelligence. Annotation information on the impact of major mutations in genomic regions is extracted from genomic databases and used to define prior information for the approach we call HBIQ. We applied HBIQ to the analysis of CNV deletions and duplications from three datasets and identified several genomic regions containing CNVs demonstrating significant deleterious effects on IQ, some of which validate previously known associations. We also show that several CNVs were identified as deleterious by HBIQ even if they have a zero pLI score, and the converse is also true. Furthermore, we show that our new model yields higher out-of-sample concordance (78%) for predicting the consequences of carrying known recurrent CNVs compared with our previous approach.

Reduced Cognitive Assessment Scores Among Individuals With Magnetic Resonance Imaging-Detected Vascular Brain Injury.

Background and Purpose- Little is known about the association between covert vascular brain injury and cognitive impairment in middle-aged populations. We investigated if scores on a cognitive screen were lower in individuals with higher cardiovascular risk, and those with covert vascular brain injury. Methods- Seven thousand five hundred forty-seven adults, aged 35 to 69 years, free of cardiovascular disease underwent a cognitive assessment using the Digital Symbol Substitution test and Montreal Cognitive Assessment, and magnetic resonance imaging (MRI) to detect covert vascular brain injury (high white matter hyperintensities, lacunar, and nonlacunar brain infarctions). Cardiovascular risk factors were quantified using the INTERHEART (A Global Study of Risk Factors for Acute Myocardial Infarction) risk score. Multivariable mixed models tested for independent determinants of reduced cognitive scores. The population attributable risk of risk factors and MRI vascular brain injury on low cognitive scores was calculated. Results- The mean age of participants was 58 (SD, 9) years; 55% were women. Montreal Cognitive Assessment and Digital Symbol Substitution test scores decreased significantly with increasing age (P<0.0001), INTERHEART risk score (P<0.0001), and among individuals with high white matter hyperintensities, nonlacunar brain infarction, and individuals with 3+ silent brain infarctions. Adjusted for age, sex, education, ethnicity covariates, Digital Symbol Substitution test was significantly lowered by 1.0 (95% CI, -1.3 to -0.7) point per 5-point cardiovascular risk score increase, 1.9 (95% CI, -3.2 to -0.6) per high white matter hyperintensities, 3.5 (95% CI, -6.4 to -0.7) per nonlacunar stroke, and 6.8 (95% CI, -11.5 to -2.2) when 3+ silent brain infarctions were present. No postsecondary education accounted for 15% (95% CI, 12-17), moderate and high levels of cardiovascular risk factors accounted for 19% (95% CI, 8-30), and MRI vascular brain injury accounted for 10% (95% CI, -3 to 22) of low test scores. Conclusions- Among a middle-aged community-dwelling population, scores on a cognitive screen were lower in individuals with higher cardiovascular risk factors or MRI vascular brain injury. Much of the population attributable risk of low cognitive scores can be attributed to lower educational attainment, higher cardiovascular risk factors, and MRI vascular brain injury.

Protein synthesis levels are increased in a subset of individuals with fragile X syndrome.

Fragile X syndrome (FXS) is a monogenic form of intellectual disability and autism spectrum disorder caused by the absence of the fragile X mental retardation protein (FMRP). In biological models for the disease, this leads to upregulated mRNA translation and as a consequence, deficits in synaptic architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here, we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here, we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those in neurons we suggest the validity of this peripheral biomarker. Our study offers a potential explanation for the comprehensive drug development program undertaken thus far yielding negative results and suggests that a significant proportion, but not all individuals with FXS, may benefit from the reduction of excessive levels of protein synthesis.

The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs.

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%-1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes.

Genotype-phenotype correlation at codon 1740 of SETD2.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

Estimating the effect size of the 15Q11.2 BP1-BP2 deletion and its contribution to neurodevelopmental symptoms: recommendations for practice.

BACKGROUND: The 15q11.2 deletion is frequently identified in the neurodevelopmental clinic. Case-control studies have associated the 15q11.2 deletion with neurodevelopmental disorders, and clinical case series have attempted to delineate a microdeletion syndrome with considerable phenotypic variability. The literature on this deletion is extensive and confusing, which is a challenge for genetic counselling. The aim of this study was to estimate the effect size of the 15q11.2 deletion and quantify its contribution to neurodevelopmental disorders. METHODS: We performed meta-analyses on new and previously published case-control studies and used statistical models trained in unselected populations with cognitive assessments. We used new (n=241) and previously published (n=150) data from a clinically referred group of deletion carriers. 15q11.2 duplications (new n=179 and previously published n=35) were used as a neutral control variant. RESULTS: The deletion decreases IQ by 4.3 points. The estimated ORs and respective frequencies in deletion carriers for intellectual disabilities, schizophrenia and epilepsy are 1.7 (3.4%), 1.5 (2%) and 3.1 (2.1%), respectively. There is no increased risk for heart malformations and autism. In the clinically referred group, the frequency and nature of symptoms in deletions are not different from those observed in carriers of the 15q11.2 duplication suggesting that most of the reported symptoms are due to ascertainment bias. CONCLUSIONS: We recommend that the deletion should be classified as 'pathogenic of mild effect size'. Since it explains only a small proportion of the phenotypic variance in carriers, it is not worth discussing in the developmental clinic or in a prenatal setting.