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De novo mutations in specific mTOR pathway genes cause brain overgrowth in the context of intellectual disability (ID). By analyzing 101 mMTOR-related genes in a large ID patient cohort and two independent population cohorts, we show that these genes modulate brain growth in health and disease. We report the mTOR activator gene RHEB as an ID gene that is associated with megalencephaly when mutated. Functional testing of mutant RHEB in vertebrate animal models indicates pathway hyperactivation with a concomitant increase in cell and head size, aberrant neuronal migration, and induction of seizures, concordant with the human phenotype. This study reveals that tight control of brain volume is exerted through a large community of mTOR-related genes. Human brain volume can be altered, by either rare disruptive events causing hyperactivation of the pathway, or through the collective effects of common alleles. The mTOR pathway is a key regulator of normal brain development. Here, the authors identify de novo mutations in RHEB, an mTOR activator protein, in patients with intellectual disability associated with megalencephaly and find a role for RHEB in regulating neuronal soma size and migration in vitro and in vivo.

Intellectual disability (ID) is a clinically and genetically heterogeneous disorder, affecting 1–3% of the general population. Although research into the genetic causes of ID has recently gained momentum, identification of pathogenic mutations that cause autosomal recessive ID (ARID) has lagged behind, predominantly due to non-availability of sizeable families. Here we present the results of exome sequencing in 121 large consanguineous Pakistani ID families. In 60 families, we identified homozygous or compound heterozygous DNA variants in a single gene, 30 affecting reported ID genes and 30 affecting novel candidate ID genes. Potential pathogenicity of these alleles was supported by co-segregation with the phenotype, low frequency in control populations and the application of stringent bioinformatics analyses. In another eight families segregation of multiple pathogenic variants was observed, affecting 19 genes that were either known or are novel candidates for ID. Transcriptome profiles of normal human brain tissues showed that the novel candidate ID genes formed a network significantly enriched for transcriptional co-expression ( P< 0.0001) in the frontal cortex during fetal development and in the temporal–parietal and sub-cortex during infancy through adulthood. In addition, proteins encoded by 12 novel ID genes directly interact with previously reported ID proteins in six known pathways essential for cognitive function ( P< 0.0001). These results suggest that disruptions of temporal parietal and sub-cortical neurogenesis during infancy are critical to the pathophysiology of ID. These findings further expand the existing repertoire of genes involved in ARID, and provide new insights into the molecular mechanisms and the transcriptome map of ID.

Key Points Whole-exome and whole-genome sequencing approaches have provided new insights into the genetics of intellectual disability (ID) and are rapidly being introduced as first-tier diagnostic assays. A genetic diagnosis can now be made in most patients with severe ID. Severe ID is mostly caused by a de novo point mutation, an insertion or deletion, or a structural genomic variation. More than 700 genes have been found to play a part in isolated ID and ID-associated disorders, and many more await discovery. New genetic research will focus on studying the role of somatic, non-coding and complex forms of inheritance in ID. An increasing number of therapeutic options is being developed for ID. In addition, preventative strategies should be aimed at reducing the number of de novo mutations in the offspring. The phenotypic heterogeneity of intellectual disability (ID) disorders has hampered studies of the underlying genetics, but major progress has been achieved by recent applications of next-generation sequencing. This Review discusses our latest understanding of ID genetics, including the identification of de novo and inherited mutations of various types, strategies for assigning disease causality to the mutations, emerging pathological mechanisms and future research directions. Genetic factors play a major part in intellectual disability (ID), but genetic studies have been complicated for a long time by the extreme clinical and genetic heterogeneity. Recently, progress has been made using different next-generation sequencing approaches in combination with new functional readout systems. This approach has provided novel insights into the biological pathways underlying ID, improved the diagnostic process and offered new targets for therapy. In this Review, we highlight the insights obtained from recent studies on the role of genetics in ID and its impact on diagnosis, prognosis and therapy. We also discuss the future directions of genetics research for ID and related neurodevelopmental disorders.

This Article was originally published under a CC BY-NC-SA 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the Article have been modified accordingly.

BackgroundAdult-onset Still’s disease (AOSD) is a rare autoinflammatory disease characterised by fever, arthritis, and multi-organ involvement. Inflammation in AOSD is mediated by interleukin (IL)−1β, as confirmed by the dramatic clinical efficacy of selective blockers of this cytokine. The genetic predisposition to this rampant IL-1-driven inflammation remains nevertheless elusive. Previous studies failed to identify associations between polymorphisms in the genes encoding IL-1 and AOSD, thus pointing at more complex genetic mechanisms. This ‘missing heritability’ cannot be adequately investigated with traditional techniques for genetic partitioning, such as GWAS, which only assess common variants and polymorphisms. Studies focusing on highly penetrant rare variants or different types of mutations (i.e. small copy-number variations; insertions/deletions) are warranted.ObjectivesWe hypothesised that genetically determined changes in IL-1-related pathways resulting in excessive IL-1β activity lead to the development of autoinflammation in AOSD. Scope of this study was to unravel the combined mutational variation of a network of IL-1-related receptors, pathways, counter-regulators, and cellular processes possibly involved in the pathogenesis of AOSD and IL-1-mediated inflammation in general.MethodsWe collected clinical, demographic, and genetic data from a large cohort of 76 AOSD patients and developed an innovative platform based on molecular inversion probes (MIP) technology for performing highly multiplexed targeted-resequencing. This allows efficient sequencing of the coding sequence of 48 genes related to the IL-1-pathway, and allows studying rare and common variants in one assay. We have also screened 500 healthy controls, and 1000s of samples with other disorders using the same assay.ResultsWe identified rare and unique (i.e. private variants) in the IL1 pathway in several individuals with AOSD. Whether any these are involved in a strong predisposition to AOSD is currently followed-up. Rare genetic variants have been identified in six IL-1-pathway ‘clusters’:Deregulated activation of the inflammasome and release of IL–1β and IL–18.IL–1 family receptors and intracellular signalling mediators.Other pro–inflammatory cytokines and receptors.Regulatory molecules, including IL–1Ra or IL–37.Cellular processes regulating production of IL–1 and IL–18 (i.e. autophagy).Production of ROS, which function as markers of cellular damage and trigger inflammation.ConclusionsUnravelling the genetic bases of inflammation in AOSD deepens our understanding of the human innate immunome. Of note, this study platform may serve for the genetic analysis of other IL-1-mediated conditions, including gout and other autoinflammatory diseases, whose genetic predisposition remains elusive. Equally important, the identification of pathways amenable to targeting with small molecules or biologics may translate into remarkable clinical implications.Disclosure of InterestNone declared

Next generation sequencing can be used to search for Mendelian disease genes in an unbiased manner by sequencing the entire protein-coding sequence, known as the exome, or even the entire human genome. Identifying the pathogenic mutation amongst thousands to millions of genomic variants is a major challenge, and novel variant prioritization strategies are required. The choice of these strategies depends on the availability of well-phenotyped patients and family members, the mode of inheritance, the severity of the disease and its population frequency. In this review, we discuss the current strategies for Mendelian disease gene identification by exome resequencing. We conclude that exome strategies are successful and identify new Mendelian disease genes in approximately 60% of the projects. Improvements in bioinformatics as well as in sequencing technology will likely increase the success rate even further. Exome sequencing is likely to become the most commonly used tool for Mendelian disease gene identification for the coming years.

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Whole-genome sequencing is used to identify genetic alterations in patients with severe intellectual disability for whom all other tests, including array and exome sequencing, returned negative results; de novo single-nucleotide and copy number variations affecting the coding region seem to be a major cause of this disorder. Gene variation in intellectual disability Intellectual disability has been shown to be linked to genetic variation but the majority of cases remain undiagnosed. This paper demonstrates the use of whole-genome sequencing to identify genetic alterations in patients with severe intellectual disability for whom all other tests, including array and exome sequencing, had returned negative results. Whole-genome sequencing of 50 patients with severe intellectual disability — and with no family history of the condition — resulted in a conclusive genetic diagnosis in 21 patients. The results suggest that de novo copy number variations and single-nucleotide variations affecting the coding region are a major cause of severe intellectual disability. Severe intellectual disability (ID) occurs in 0.5% of newborns and is thought to be largely genetic in origin 1 , 2 . The extensive genetic heterogeneity of this disorder requires a genome-wide detection of all types of genetic variation. Microarray studies and, more recently, exome sequencing have demonstrated the importance of de novo copy number variations (CNVs) and single-nucleotide variations (SNVs) in ID, but the majority of cases remain undiagnosed 3 , 4 , 5 , 6 . Here we applied whole-genome sequencing to 50 patients with severe ID and their unaffected parents. All patients included had not received a molecular diagnosis after extensive genetic prescreening, including microarray-based CNV studies and exome sequencing. Notwithstanding this prescreening, 84 de novo SNVs affecting the coding region were identified, which showed a statistically significant enrichment of loss-of-function mutations as well as an enrichment for genes previously implicated in ID-related disorders. In addition, we identified eight de novo CNVs, including single-exon and intra-exonic deletions, as well as interchromosomal duplications. These CNVs affected known ID genes more frequently than expected. On the basis of diagnostic interpretation of all de novo variants, a conclusive genetic diagnosis was reached in 20 patients. Together with one compound heterozygous CNV causing disease in a recessive mode, this results in a diagnostic yield of 42% in this extensively studied cohort, and 62% as a cumulative estimate in an unselected cohort. These results suggest that de novo SNVs and CNVs affecting the coding region are a major cause of severe ID. Genome sequencing can be applied as a single genetic test to reliably identify and characterize the comprehensive spectrum of genetic variation, providing a genetic diagnosis in the majority of patients with severe ID.

Joris Veltman, Han Brunner and colleagues report results of a family based exome sequencing study of ten individuals with unexplained mental retardation. They identified and validated de novo mutations in nine genes, six of which are likely to be pathogenic based on functional criteria, suggesting an important role for de novo point mutations in the etiology of unexplained mental retardation. The per-generation mutation rate in humans is high. De novo mutations may compensate for allele loss due to severely reduced fecundity in common neurodevelopmental and psychiatric diseases, explaining a major paradox in evolutionary genetic theory. Here we used a family based exome sequencing approach to test this de novo mutation hypothesis in ten individuals with unexplained mental retardation. We identified and validated unique non-synonymous de novo mutations in nine genes. Six of these, identified in six different individuals, are likely to be pathogenic based on gene function, evolutionary conservation and mutation impact. Our findings provide strong experimental support for a de novo paradigm for mental retardation. Together with de novo copy number variation, de novo point mutations of large effect could explain the majority of all mental retardation cases in the population.

Polycystic livers are seen in the rare inherited disorder isolated polycystic liver disease (PCLD) and are recognized as the most common extrarenal manifestation in autosomal dominant polycystic kidney disease. Hepatic cystogenesis is characterized by progressive proliferation of cholangiocytes, ultimately causing hepatomegaly. Genetically, polycystic liver disease is a heterogeneous disorder with incomplete penetrance and caused by mutations in PRKCSH , SEC63 , PKD1 , or PKD2 . Genome-wide SNP typing and Sanger sequencing revealed no pathogenic variants in hitherto genes in an extended PCLD family. We performed whole-exome sequencing of DNA samples from two members. A heterozygous variant c.3562C > T located at a highly conserved amino acid position (p.R1188W) in the low density lipoprotein receptor-related protein 5 (LRP5) gene segregated with the disease (logarithm of odds score, 4.62) but was not observed in more than 1,000 unaffected individuals. Screening of LRP5 in a PCLD cohort identified three additional mutations in three unrelated families with polycystic livers (p.V454M, p.R1529S, and p.D1551N), again all undetected in controls. All variants were predicted to be damaging with profound structural effects on LRP5 protein domains. Liver cyst tissue and normal hepatic tissue samples from patients and controls showed abundant LRP5 expression by immunohistochemistry. Functional activity analyses indicated that mutant LRP5 led to reduced wingless signal activation. In conclusion, we demonstrate that germ-line LRP5 missense mutations are associated with hepatic cystogenesis. The findings presented in this study link the pathophysiology of PCLD to deregulation of the canonical wingless signaling pathway.