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Voltage-gated calcium (CaV) channels form three subfamilies (CaV1–3). The CaV1 and CaV2 channels are heteromeric, consisting of an α1 pore-forming subunit, associated with auxiliary CaVβ and α2δ subunits. The α2δ subunits are encoded in mammals by four genes, CACNA2D1–4. They play important roles in trafficking and function of the CaV channel complexes. Here we report biallelic variants in CACNA2D1, encoding the α2δ-1 protein, in two unrelated individuals showing a developmental and epileptic encephalopathy. Patient 1 has a homozygous frameshift variant c.818_821dup/p.(Ser275Asnfs*13) resulting in nonsense-mediated mRNA decay of the CACNA2D1 transcripts, and absence of α2δ-1 protein detected in patient-derived fibroblasts. Patient 2 is compound heterozygous for an early frameshift variant c.13_23dup/p.(Leu9Alafs*5), highly probably representing a null allele and a missense variant c.626G>A/p.(Gly209Asp). Our functional studies show that this amino-acid change severely impairs the function of α2δ-1 as a calcium channel subunit, with strongly reduced trafficking of α2δ-1G209D to the cell surface and a complete inability of α2δ-1G209D to increase the trafficking and function of CaV2 channels. Thus, biallelic loss-of-function variants in CACNA2D1 underlie the severe neurodevelopmental disorder in these two patients. Our results demonstrate the critical importance and non-interchangeability of α2δ-1 and other α2δ proteins for normal human neuronal development.

The major spliceosome mediates pre-mRNA splicing by recognizing the highly conserved sequences at the 5′ and 3′ splice sites and the branch point. More than 150 proteins participate in the splicing process and are organized in the spliceosomal A, B, and C complexes. FRA10AC1 is a peripheral protein of the spliceosomal C complex and its ortholog in the green alga facilitates recognition or interaction with splice sites. We identified biallelic pathogenic variants in FRA10AC1 in five individuals from three consanguineous families. The two unrelated Patients 1 and 2 with loss-of-function variants showed developmental delay, intellectual disability, and no speech, while three siblings with the c.494_496delAAG (p.Glu165del) variant had borderline to mild intellectual disability. All patients had microcephaly, hypoplasia or agenesis of the corpus callosum, growth retardation, and craniofacial dysmorphism. FRA10AC1 transcripts and proteins were drastically reduced or absent in fibroblasts of Patients 1 and 2. In a heterologous expression system, the p.Glu165del variant impacts intrinsic stability of FRA10AC1 but does not affect its nuclear localization. By co-immunoprecipitation, we found ectopically expressed HA-FRA10AC1 in complex with endogenous DGCR14, another component of the spliceosomal C complex, while the splice factors CHERP, NKAP, RED, and SF3B2 could not be co-immunoprecipitated. Using an in vitro splicing reporter assay, we did not obtain evidence for FRA10AC1 deficiency to suppress missplicing events caused by mutations in the highly conserved dinucleotides of 5′ and 3′ splice sites in an in vitro splicing assay in patient-derived fibroblasts. Our data highlight the importance of specific peripheral spliceosomal C complex proteins for neurodevelopment. It remains possible that FRA10AC1 may have other and/or additional cellular functions, such as coupling of transcription and splicing reactions.

Contains fulltext : 237838.pdf (Publisher’s version ) (Closed access) The MCM2-7 helicase is a heterohexameric complex with essential roles as part of both the pre-replication and pre-initiation complexes in the early stages of DNA replication. Meier-Gorlin syndrome, a rare primordial dwarfism, is strongly associated with disruption to the pre-replication complex, including a single case described with variants in MCM5. Conversely, a biallelic pathogenic variant in MCM4 underlies immune deficiency with growth retardation, features also seen in individuals with pathogenic variants in other pre-initiation complex encoding genes such as GINS1, MCM10, and POLE. Through exome and chromium genome sequencing, supported by functional studies, we identify biallelic pathogenic variants in MCM7 and a strong candidate biallelic pathogenic variant in MCM3. We confirm variants in MCM7 are deleterious and through interfering with MCM complex formation, impact efficiency of S phase progression. The associated phenotypes are striking; one patient has typical Meier-Gorlin syndrome, whereas the second case has a multi-system disorder with neonatal progeroid appearance, lipodystrophy and adrenal insufficiency. We provide further insight into the developmental complexity of disrupted MCM function, highlighted by two patients with a similar variant profile in MCM7 but disparate clinical features. Our results build on other genetic findings linked to disruption of the pre-replication and pre-initiation complexes, and the replisome, and expand the complex clinical genetics landscape emerging due to disruption of DNA replication.

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulphated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report biallelic pathogenic variants in the HS2ST1 gene in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals showed an increased length and concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a novel recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal and renal development.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b., Author summary Neurodevelopmental disorders encompass a broad spectrum of neurological and psychiatric conditions and are caused by mutations in many different genes. For example, mutations in genes encoding voltage-gated calcium channels have been linked to various diseases of the nervous system in humans and mice. Voltage-gated calcium channels are critical regulators of the synaptic communication between neurons, of processes involved in learning and memory, and of activity-dependent gene expression. Here we report a disease-associated mutation on both copies of the CACNB4 gene encoding an auxiliary β4 subunit of the chief presynaptic calcium channel in the brain. Two siblings with a severe neurodevelopmental disorder carry the homozygous CACNB4 mutation causing an amino acid substitution known to disrupt the folding of the calcium channel β4 subunit. We demonstrate that this amino acid change abolished the incorporation of the β4 subunit into channel complexes in the synapse, as well as β4’s ability to translocate into the cell nucleus, and to complex with α1 channel subunits and a neuronal scaffolding protein. The combined evidence from our genetic and functional analysis suggests that dysfunction of both β4 subunit channel and non-channel functions underlies the severe neurological phenotype in the two siblings. We therefore identified CACNB4 as a neurodevelopmental disease gene.

Spondyloepimetaphyseal dysplasias (SEMD) are a group of genetically heterogeneous skeletal disorders characterized by abnormal vertebral bodies and epimetaphyseal abnormalities. We investigated two families with a new SEMD type with one proband each. They showed mild facial dysmorphism, flat vertebral bodies (platyspondyly), large epiphyses, metaphyseal dysplasia, and hallux valgus as common clinical features. By trio-exome sequencing, the homozygous missense variant c.797G>A/p.(Cys266Tyr) in PISD was found in both affected individuals. Based on exome data analyses for homozygous regions, the two patients shared a single homozygous block on chromosome 22 including PISD, indicating their remote consanguinity. PISD encodes phosphatidylserine (PS) decarboxylase that is localized in the inner mitochondrial membrane and catalyzes the decarboxylation of PS to phosphatidylethanolamine (PE) in mammalian cells. PE occurs at high abundance in mitochondrial membranes. Patient-derived fibroblasts showed fragmented mitochondrial morphology. Treatment of patient cells with MG-132 or staurosporine to induce activation of the intrinsic apoptosis pathway revealed significantly decreased cell viability with increased caspase-3 and caspase-7 activation. Remarkably, ethanolamine (Etn) supplementation largely restored cell viability and enhanced apoptosis in MG-132-stressed patient cells. Our data demonstrate that the biallelic hypomorphic PISD variant p.(Cys266Tyr) is associated with a novel SEMD form, which may be treatable with Etn administration.

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS., Author summary Noonan syndrome (NS) belongs to the RASopathies, a group of developmental diseases caused by mutations in genes encoding RAS-MAPK pathway components. Germline mutations in RIT1 have been identified in NS. RIT1 belongs to the RAS superfamily, however, the cellular function of RIT1 remains elusive. We show that RIT1 binds p21-activated kinase 1 (PAK1), an effector of the RHO GTPases RAC1 and CDC42, which are important regulators of cytoskeletal dynamics. NS-associated RIT1 mutants enhance complex formation between RIT1, RAC1/CDC42 and PAK1. Expression of wild-type or mutant forms of RIT1 caused loss of stress fibers and mature focal adhesions and enhanced cell motility. Our data suggest that dysfunction in actin dynamics is a novel aspect in the pathophysiology of RASopathies.

Previous studies have shown that in the rat, the colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is only absorbed to a limited extent in the small intestines and that a major fraction of unmetabolised PhIP reaches the colon. Moreover, PhIP is extensively metabolised when incubated with human stool samples to a major derivative, 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido [3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1). In the present study, the uptake and transport of PhIP-M1 in Ussing chamber experiments, its cytotoxicity in the different segments of the Fischer 344 rat gut and its transforming potential in the BALB/c 3T3 cell transformation assay were analysed. At the most, 10-20% of the PhIP-M1 amount added to the mucosal compartment of the Ussing chambers per segment were absorbed within 90min. Therefore, the amount of PhIP-M1 detected in the tissues as well as in the serosal compartment of the Ussing chambers was extremely low. Moreover, human-relevant concentrations of PhIP-M1 were not cytotoxic and did not induce the malignant transformation of BALB/c 3T3 cells. In conclusion, even if one would assume that 100% of the daily amount of PhIP ingested by a human being is converted into PhIP-M1 in the colon, this concentration most probably would not lead to cytotoxicity and/or carcinogenicity in the colorectal mucosa.

The ADP-ribosyltransferase C3 exoenzyme from C. botulinum selectively inactivates Rho and is therefore often used as an inhibitor for investigations on Rho signaling. Previous studies of our group revealed that C3 inhibited cell proliferation in HT22 cells accompanied by increased transcriptional activities of Sp1 and c-Jun and reduced levels of cyclin D1, p21 and phosphorylated p38. By use of a p38α-deficient and a p38α-expressing control cell line, the impact of p38 on C3-mediated inhibition of cell proliferation and alterations on MAPK signaling was studied by growth kinetic experiments and Western blot analyses. The cell growth of p38α-expressing cells was impaired by C3, while the p38α-deficient cells did not exhibit any C3-induced effect. The activity of the MKK3/6-p38 MAPK signaling cascade as well as the phosphorylation of c-Jun and JNK was reduced by C3 exclusively in the presence of p38α. Moreover, the activity of upstream MAPKKK TAK1 was lowered in the p38α-expressing cells. These results indicated a resistance of p38α-deficient cells to C3-mediated inhibition of cell growth. This anti-proliferative effect was highly associated with the decreased activity of c-Jun and upstream p38 and JNK MAPK signaling as a consequence of the absence of p38α in these cells.

C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

Heterocyclic aromatic amines such as PHIP are formed during the heat processing of food. PHIP undergoes bacterial metabolism leading to 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PHIP-M1) as main metabolite. We developed an LC-MS method with automated sample preparation by online-solid-phase-extraction for the simultaneous quantification of PHIP and its mammalian and bacterial metabolites N-hydroxy-PHIP, 4-OH-PHIP and PHIP-M1 in biological samples. The method was used to investigate the transport of PHIP-M1 through a Caco-2 cell monolayer. The experiments show that PHIP-M1 rapidly crosses the cell monolayer and that PHIP-M1 is a substrate for P-glycoprotein and the multiple drug resistance 2 transporter. The intestinal absorption of PHIP-M1 is comparable with that of PHIP and a moderate to high bioavailability has to be expected. Thus, not only the human metabolites of PHIP but also the bacterial metabolite PHIP-M1 formed in the gut could contribute to the toxic effects of PhIP.

C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors. Electronic supplementary material The online version of this article (doi:10.1007/s00210-016-1270-2) contains supplementary material, which is available to authorized users.