Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclinical seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clinical presentation in the two families, we hypothesized that the observed phenotype was due to mutations in the same gene, and we performed exome sequencing in three affected individuals. Analysis of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease association was confirmed by cosegregation analysis in additional family members. Screening of 68 additional unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one additional subject with compound heterozygous mutations of SLC13A5 and a similar clinical presentation as the index subjects. Mutations affected key residues for sodium binding, which is critical for citrate transport. These findings underline the value of careful clinical characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metabolism in epilepsy.

Intellectual Disability (ID) is characterized by deficits in intellectual functions such as reasoning, problem-solving, planning, abstract thinking, judgment, and learning. As new avenues are emerging for treatment of genetically determined ID (such as Down's syndrome or Fragile X syndrome), it is necessary to identify objective reliable and sensitive outcome measures for use in clinical trials.

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.

Mutations in mediator of RNA polymerase II transcription subunit 12 homolog (MED12, OMIM 300188) cause X-linked intellectual disability (XLID) disorders including FG, Lujan, and Ohdo syndromes. The Gli3-dependent Sonic Hedgehog (SHH) signaling pathway has been implicated in the original FG syndrome and Lujan syndrome. How are SHH-signaling defects related to the complex clinical phenotype of MED12-associated XLID syndromes are not fully understood.

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.

Genomics features with similar genome-wide distributions are generally hypothesized to be functionally related, for example, colocalization of histones and transcription start sites indicate chromatin regulation of transcription factor activity. Therefore, statistical algorithms to perform spatial, genome-wide correlation among genomic features are required.

We have identified TUBGCP4 variants in individuals with autosomal-recessive microcephaly and chorioretinopathy. Whole-exome sequencing performed on one family with two affected siblings and independently on another family with one affected child revealed compound-heterozygous mutations in TUBGCP4. Subsequent Sanger sequencing was performed on a panel of individuals from 12 French families affected by microcephaly and ophthalmic manifestations, and one other individual was identified with compound-heterozygous mutations in TUBGCP4. One synonymous variant was common to all three families and was shown to induce exon skipping; the other mutations were frameshift mutations and a deletion. TUBGCP4 encodes γ-tubulin complex protein 4, a component belonging to the γ-tubulin ring complex (γ-TuRC) and known to regulate the nucleation and organization of microtubules. Functional analysis of individual fibroblasts disclosed reduced levels of the γ-TuRC, altered nucleation and organization of microtubules, abnormal nuclear shape, and aneuploidy. Moreover, zebrafish treated with morpholinos against tubgcp4 were found to have reduced head volume and eye developmental anomalies with chorioretinal dysplasia. In summary, the identification of TUBGCP4 mutations in individuals with microcephaly and a spectrum of anomalies in eye development, particularly photoreceptor anomalies, provides evidence of an important role for the γ-TuRC in brain and eye development.

Waardenburg syndrome (WS) is an auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair and skin. Depending on additional symptoms, WS is classified into four subtypes, WS1-WS4. Absence of additional features characterizes WS2. The association of facial dysmorphic features defines WS1 and WS3, whereas the association with Hirschsprung disease (aganglionic megacolon) characterizes WS4, also called "Waardenburg-Hirschsprung disease." Mutations within the genes MITF and SNAI2 have been identified in WS2, whereas mutations of EDN3, EDNRB, and SOX10 have been observed in patients with WS4. However, not all cases are explained at the molecular level, which raises the possibility that other genes are involved or that some mutations within the known genes are not detected by commonly used genotyping methods. We used a combination of semiquantitative fluorescent multiplex polymerase chain reaction and fluorescent in situ hybridization to search for SOX10 heterozygous deletions. We describe the first characterization of SOX10 deletions in patients presenting with WS4. We also found SOX10 deletions in WS2 cases, making SOX10 a new gene of WS2. Interestingly, neurological phenotypes reminiscent of that observed in WS4 (PCWH syndrome [peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, WS, and Hirschsprung disease]) were observed in some WS2-affected patients with SOX10 deletions. This study further characterizes the molecular complexity and the close relationship that links the different subtypes of WS.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in RNU4ATAC, transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning RNU4ATAC-mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Through linkage analysis, homozygosity mapping, and exome sequencing in four unrelated families affected by lethal AMC, we identified biallelic mutations in GLDN in the affected individuals. GLDN encodes gliomedin, a secreted cell adhesion molecule involved in the formation of the nodes of Ranvier. Transmission electron microscopy of the sciatic nerve from one of the affected individuals showed a marked lengthening defect of the nodes. The GLDN mutations found in the affected individuals abolish the cell surface localization of gliomedin and its interaction with its axonal partner, neurofascin-186 (NF186), in a cell-based assay. The axoglial contact between gliomedin and NF186 is essential for the initial clustering of Na+ channels at developing nodes. These results indicate a major role of gliomedin in node formation and the development of the peripheral nervous system in humans. These data indicate that mutations of GLDN or CNTNAP1 (MIM: 616286), encoding essential components of the nodes of Ranvier and paranodes, respectively, lead to inherited nodopathies, a distinct disease entity among peripheral neuropathies.

To evaluate the role that chromosomal micro-rearrangements play in patients with both corpus callosum abnormality and intellectual disability, we analyzed copy number variations (CNVs) in patients with corpus callosum abnormality/intellectual disability STUDY DESIGN: We screened 149 patients with corpus callosum abnormality/intellectual disability using Illumina SNP arrays.

KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Although the role of typical Rho GTPases and other Rho-linked proteins in synaptic plasticity and cognitive function and dysfunction is widely acknowledged, the role of atypical Rho GTPases (such as RHOBTB2) in neurodevelopment has barely been characterized. We have now identified de novo missense variants clustering in the BTB-domain-encoding region of RHOBTB2 in ten individuals with a similar phenotype, including early-onset epilepsy, severe intellectual disability, postnatal microcephaly, and movement disorders. Three of the variants were recurrent. Upon transfection of HEK293 cells, we found that mutant RHOBTB2 was more abundant than the wild-type, most likely because of impaired degradation in the proteasome. Similarly, elevated amounts of the Drosophila ortholog RhoBTB in vivo were associated with seizure susceptibility and severe locomotor defects. Knockdown of RhoBTB in the Drosophila dendritic arborization neurons resulted in a decreased number of dendrites, thus suggesting a role of RhoBTB in dendritic development. We have established missense variants in the BTB-domain-encoding region of RHOBTB2 as causative for a developmental and epileptic encephalopathy and have elucidated the role of atypical Rho GTPase RhoBTB in Drosophila neurological function and possibly dendrite development.

Phelan-McDermid syndrome (PMS) is characterized by a variety of clinical symptoms with heterogeneous degrees of severity, including intellectual disability (ID), absent or delayed speech, and autism spectrum disorders (ASD). It results from a deletion of the distal part of chromosome 22q13 that in most cases includes the SHANK3 gene. SHANK3 is considered a major gene for PMS, but the factors that modulate the severity of the syndrome remain largely unknown. In this study, we investigated 85 patients with different 22q13 rearrangements (78 deletions and 7 duplications). We first explored the clinical features associated with PMS, and provide evidence for frequent corpus callosum abnormalities in 28% of 35 patients with brain imaging data. We then mapped several candidate genomic regions at the 22q13 region associated with high risk of clinical features, and suggest a second locus at 22q13 associated with absence of speech. Finally, in some cases, we identified additional clinically relevant copy-number variants (CNVs) at loci associated with ASD, such as 16p11.2 and 15q11q13, which could modulate the severity of the syndrome. We also report an inherited SHANK3 deletion transmitted to five affected daughters by a mother without ID nor ASD, suggesting that some individuals could compensate for such mutations. In summary, we shed light on the genotype-phenotype relationship of patients with PMS, a step towards the identification of compensatory mechanisms for a better prognosis and possibly treatments of patients with neurodevelopmental disorders.

Autism spectrum disorders (ASDs) are characterized by deficits in three core behavioral domains: reciprocal social interactions, communication, and restricted interests and/or repetitive behaviors. Several hundreds of risk genes for autism have been identified, however, it remains a challenge to associate these genes with specific core behavioral deficits. In multiplex autism families, affected sibs often show significant differences in severity of individual core phenotypes. We hypothesize that a higher mutation burden contributes to a larger difference in the severity of specific core phenotypes between affected sibs. We tested this hypothesis on social behavioral deficits in autism. We sequenced synaptome genes (n = 1,886) in affected male sib-pairs (n = 274) in families from the Autism Genetics Research Exchange (AGRE) and identified rare (MAF ≤ 1%) and predicted functional variants. We selected affected sib-pairs with a large (≥10; n = 92 pairs) or a small (≤4; n = 108 pairs) difference in total cumulative Autism Diagnostic Interview-Revised (ADI-R) social scores (SOCT_CS). We compared burdens of unshared variants present only in sibs with severe social deficits and found a higher burden in SOCT_CS≥10 compared to SOCT_CS ≤ 4 (SOCT_CS≥10: 705.1 ± 16.2; SOCT_CS ≤ 4, 668.3 ± 9.0; p = 0.025). Unshared SOCT_CS≥10 genes only in sibs with severe social deficits are significantly enriched in the SFARI gene set. Network analyses of these genes using InWeb_IM, molecular signatures database (MSigDB), and GeNetMeta identified enrichment for phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) (Enrichment Score [eScore] p value = 3.36E-07; n = 8 genes) and Nerve growth factor (NGF) (eScore p value = 8.94E-07; n = 9 genes) networks. These studies support a key role for these signaling networks in social behavioral deficits and present a novel approach to associate risk genes and signaling networks with core behavioral domains in autism.

Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

LIMK2 is involved in neuronal functions by regulating actin dynamics. Different isoforms of LIMK2 are described in databanks. LIMK2a and LIMK2b are the most characterized. A few pieces of evidence suggest that LIMK2 isoforms might not have overlapping functions. In this study, we focused our attention on a less studied human LIMK2 isoform, LIMK2-1. Compared to the other LIMK2 isoforms, LIMK2-1 contains a supplementary C-terminal phosphatase 1 inhibitory domain (PP1i). We found out that this isoform was hominidae-specific and showed that it was expressed in human fetal brain and faintly in adult brain. Its coding sequence was sequenced in 173 patients with sporadic non-syndromic intellectual disability (ID), and we observed an association of a rare missense variant in the PP1i domain (rs151191437, p.S668P) with ID. Our results also suggest an implication of LIMK2-1 in neurite outgrowth and neurons arborization which appears to be affected by the p.S668P variation. Therefore our results suggest that LIMK2-1 plays a role in the developing brain, and that a rare variation of this isoform is a susceptibility factor in ID.

Laminin α2 deficient congenital muscular dystrophy, caused by mutations in the LAMA2 gene, is characterized by early muscle weakness associated with abnormal white matter signal on cerebral MRI.