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Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioral problems and developmental delay. 80% of Dravet syndrome patients have a mutation inSCN1A, encoding NaV1.1. Milder clinical phenotypes, such as GEFS+(generalized epilepsy with febrile seizures plus), can also arise fromSCN1Amutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. Both this clinical and genetic heterogeneity add to the difficulties of predicting disease progression and tailored prescription of anti-seizure medication. A better understanding of the neuropathology of differentSCN1Amutations, might give insight in differentiating the expected clinical phenotype and best fit treatment choice. Initially it was recognized that loss of Na+-current in inhibitory neurons specifically resulted in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated, and might depend on the patient clinical phenotype or the specific mutation inSCN1A.To examine the genotype-phenotype correlations ofSCN1Amutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harboring different mutations inSCN1A, plus a family where the same mutation leads to both GEFS+ and Dravet syndrome.We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponded to seizurogenic network prediction patterns elicited by proconvulsive compounds. We find that excitatory neuronal networks were differently affected, dependent on the type of SCN1Amutation, but not on clinical severity. Specifically, pore domain mutations could be distinguished from voltage sensing domain mutations. Furthermore, all patients showed aggravated neuronal network responses upon febrile temperatures. While the basal neuronal network phenotypes could not be distinguished based on patient clinical severity, retrospective drug screening revealed that anti-seizure medication only affected GEFS+ patient-, but not Dravet patient-derived neuronal networks in a patient specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.HighlightsHuman stem cell derived excitatory neurons are affected by mutations inSCN1Aand display mutation-specific, but not clinical phenotype specific, neuronal network phenotypesThe neuronal network phenotype we describe corresponds to seizurogenic network prediction patterns in vitroExcitatory neuronal networks respond to Dravet syndrome clinically relevant triggers, like febrile temperatures and Dravet-contraindicated ASM CarbamazepineRetrospective drug screening revealed that GEFS+ neuronal networks, but not Dravet neuronal networks respond to ASM in a patient-specific and clinical relevant manner

SummaryHeterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, were shown to cause a novel neurodevelopmental syndrome and increase the risk for schizophrenia. We generated excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-) using CRISPR/Cas9. Our data show that SETD1A haploinsufficiency resulted in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype was primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling revealed perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identified specific changes in the cAMP/PKA pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway we were able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034-429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co-segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub-isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.

Item does not contain fulltext The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.

Background: Endocrine-cerebro-osteodysplasia (ECO) syndrome [MIM:612651] caused by a recessive mutation (p.R272Q) in Intestinal cell kinase (ICK) shows significant clinical overlap with ciliary disorders. Similarities are strongest between ECO syndrome, the Majewski and Mohr-Majewski short-rib thoracic dysplasia (SRTD) with polydactyly syndromes, and hydrolethalus syndrome. In this study, we present a novel homozygous ICK mutation in a fetus with ECO syndrome and compare the effect of this mutation with the previously reported ICK variant on ciliogenesis and cilium morphology. Results: Through homozygosity mapping and whole-exome sequencing, we identified a second variant (c.358G > T; p.G120C) in ICK in a Turkish fetus presenting with ECO syndrome. In vitro studies of wild-type and mutant mRFP-ICK (p.G120C and p.R272Q) revealed that, in contrast to the wild-type protein that localizes along the ciliary axoneme and/or is present in the ciliary base, mutant proteins rather enrich in the ciliary tip. In addition, immunocytochemistry revealed a decreased number of cilia in ICK p.R272Q-affected cells. Conclusions: Through identification of a novel ICK mutation, we confirm that disruption of ICK causes ECO syndrome, which clinically overlaps with the spectrum of ciliopathies. Expression of ICK-mutated proteins result in an abnormal ciliary localization compared to wild-type protein. Primary fibroblasts derived from an individual with ECO syndrome display ciliogenesis defects. In aggregate, our findings are consistent with recent reports that show that ICK regulates ciliary biology in vitro and in mice, confirming that ECO syndrome is a severe ciliopathy., Dutch Kidney Foundation (KOUNCIL consortium project CP11.18 to HHA) and the Netherlands Organization for Health Research and Development (ZonMW Veni-91613008 to HHA) and the Netherlands Organization for Scientific research (NWO Vici-016130664 to RR). This work was also funded by a Strategic Positioning Fund on Genetic Orphan Diseases from A*STAR, Singapore, and the Scientific and Technological Research Council of Turkey (TÜBİTAK) (TUBITAK) by 112S398 to HK (E-RARE network CRANIRARE-2)

Item does not contain fulltext Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.

Contains fulltext : 167699.pdf (Publisher’s version ) (Open Access) Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.

Background Mainzer–Saldino syndrome (MZSDS) is a skeletal ciliopathy and part of the short-rib thoracic dysplasia (SRTD) group of ciliary disorders. The main characteristics of MZSDS are short limbs, mild narrow thorax, blindness, and renal failure. Thus far, variants in two genes are associated with MZSDS: IFT140, and IFT172. In this study, we describe a 1-year-old girl presenting with mild skeletal abnormalities, Leber congenital amaurosis, and bilateral hearing difficulties. For establishing an accurate diagnosis, we combined clinical, molecular, and functional analyses. Methods We performed diagnostic whole-exome sequencing (WES) analysis to determine the genetic cause of the disease and analyzed two gene panels, containing all currently known genes in vision disorders, and in hearing impairment. Upon detection of the likely causative variants, ciliary phenotyping was performed in patient urine-derived renal epithelial cells (URECs) and rescue experiments were performed in CRISPR/Cas9-derived Ift140 knock out cells to determine the pathogenicity of the detected variants in vitro. Cilium morphology, cilium length, and intraflagellar transport (IFT) were evaluated by immunocytochemistry. Results Diagnostic WES revealed two novel compound heterozygous variants in IFT140, encoding IFT140. Thorough investigation of WES data did not reveal any variants in candidate genes associated with hearing impairment. Patient-derived URECs revealed an accumulation of IFT-B protein IFT88 at the ciliary tip in 41% of the cells indicative of impaired retrograde IFT, while this was absent in cilia from control URECs. Furthermore, transfection of CRISPR/Cas9-derived Ift140 knock out cells with an IFT140 construct containing the patient mutation p.Tyr923Asp resulted in a significantly higher percentage of IFT88 tip accumulation than transfection with the wild-type IFT140 construct. Conclusions By combining the clinical, genetic, and functional data from this study, we could conclude that the patient has SRTD9, also called Mainzer–Saldino syndrome, caused by variants in IFT140. We suggest the possibility that variants in IFT140 may underlie hearing impairment. Moreover, we show that urine provides an excellent source to obtain patient-derived cells in a non-invasive manner to study the pathogenicity of variants detected by genetic testing. Electronic supplementary material The online version of this article (10.1186/s13630-018-0055-2) contains supplementary material, which is available to authorized users.