Case report: Compound heterozygous NUP85 variants cause autosomal recessive primary microcephaly

Nucleoporin (NUP) 85 is a member of the Y-complex of nuclear pore complex (NPC) that is key for nucleocytoplasmic transport function, regulation of mitosis, transcription, and chromatin organization. Mutations in various nucleoporin genes have been linked to several human diseases. Among them, NUP85 was linked to childhood-onset steroid-resistant nephrotic syndrome (SRNS) in four affected individuals with intellectual disability but no microcephaly. Recently, we broaden the phenotype spectrum of NUP85-associated disease by reporting NUP85 variants in two unrelated individuals with primary autosomal recessive microcephaly (MCPH) and Seckel syndrome (SCKS) spectrum disorders (MCPH-SCKS) without SRNS. In this study, we report compound heterozygous NUP85 variants in an index patient with only MCPH phenotype, but neither Seckel syndrome nor SRNS was reported. We showed that the identified missense variants cause reduced cell viability of patient-derived fibroblasts. Structural simulation analysis of double variants is predicted to alter the structure of NUP85 and its interactions with neighboring NUPs. Our study thereby further expands the phenotypic spectrum of NUP85-associated human disorder and emphasizes the crucial role of NUP85 in the brain development and function

Synthesis and Characterization of Silver and Gold Nanoparticles Using Aqueous Extract of Seaweed, Turbinaria conoides,

Silver and gold nanoparticles were synthesized using an aqueous extract of the seaweed Turbinaria conoides and their antibiofilm activity against marine biofilm forming bacteria is reported here. The UV-Vis spectra showed the characteristics SPR absorption band for Ag NPs at 421 and for Au NPs at 538 nm. Further, the synthesized nanoparticles were characterized using FT-IR, XRD, FESEM, EDX, and HRTEM analysis. Spherical and triangular nanostructures of the Ag and Au nanoparticles were observed between the size ranges of 2–17 nm and 2–19 nm, respectively. The synthesized Ag NPs are efficient in controlling the bacterial biofilm formation; however, Au NPs did not show any remarkable antibiofilm activity. The maximum zone of inhibition was recorded against E. coli (17.6±0.42 mm), followed by Salmonella sp., S. liquefaciens, and A. hydrophila. The macrotube dilution method inferred the MIC (20–40 µL mL−1) and MBC (40–60 µL mL−1) of Ag NPs. The CLSM images clearly showed the weak adherence and disintegrating biofilm formation of marine biofilm bacterial strains treated with Ag NPs. The Artemia cytotoxicity assay recorded the LC50 value of 88.914±5.04 µL mL−1. Thus the present study proved the efficiency of Ag NPs as a potent antimicrofouling agent and became the future perspective for the possible usage in the biofouling related issues in the aquaculture installations and other marine systems

PTRH2 is Necessary for Purkinje Cell Differentiation and Survival and its Loss Recapitulates Progressive Cerebellar Atrophy and Ataxia Seen in IMNEPD Patients

Hom ozygous variants in the peptidyl-tRNA hydrolase 2 gene (PTRH2) cause infantile-onset multisystem neurologic, endocrine, and pancreatic disease. The objective is to delineate the mechanisms underlying the core cerebellar phenotype in this disease. For this, we generated constitutive (Ptrh2LoxPxhCMVCre, Ptrh2−/− mice) and Purkinje cell (PC) specific (Ptrh2LoxPxPcp2Cre, Ptrh2ΔPCmice) Ptrh2 mutant mouse models and investigated the effect of the loss of Ptrh2 on cerebellar development. We show that Ptrh2−/− knockout mice had severe postnatal runting and lethality by postnatal day 14. Ptrh2ΔPC PC specific knockout mice survived until adult age; however, they showed progressive cerebellar atrophy and functional cerebellar deficits with abnormal gait and ataxia. PCs of Ptrh2ΔPC mice had reduced cell size and density, stunted dendrites, and lower levels of ribosomal protein S6, a readout of the mammalian target of rapamycin pathway. By adulthood, there was a marked loss of PCs. Thus, we identify a cell autonomous requirement for PTRH2 in PC maturation and survival. Loss of PTRH2 in PCs leads to downregulation of the mTOR pathway and PC atrophy. This suggests a molecular mechanism underlying the ataxia and cerebellar atrophy seen in patients with PTRH2 mutations leading to infantile-onset multisystem neurologic, endocrine, and pancreatic disease

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain- hindbrain malformation

Abstract Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain- hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder. Author summary During brain development, localized gene expression is crucial for the formation and function of specific brain regions. Various groups of proteins are known to regulate segmentation through controlled gene expression, among them, the Rho GTPase regulator family. In this study, we identified a frameshift mutation in the Rho guanine nucleotide exchange factor 2 gene (ARHGEF2) in two children presenting with intellectual disability, mild microcephaly, and a midbrain- hindbrain malformation. This phenotype is also observed in Arhgef2 mutant mice, highlighting the importance of ARHGEF2 across development of distinct mammalian species. We show that loss of Arhgef2 affects neurogenesis and also cell migration. In addition, we extended the current knowledge of ARHGEF2 expression and its role in early central nervous system development, with special reference to the formation of the precerebellar system. In addition to extensive literature on ARHGEF2, we now provide evidence for its significant role in neuronal migration in brain development and link the gene to human neurodevelopmental disease

Proteome changes in autosomal recessive primary microcephaly

Background/aim: : Autosomal recessive primary microcephaly (MCPH) is a rare and genetically heterogeneous group of disorders characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. MCPH3 is caused by biallelic variants in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2. In the corresponding Cdk5rap2 mutant or Hertwig's anemia mouse model, congenital microcephaly as well as defects in the hematopoietic system, germ cells and eyes have been reported. The reduction in brain volume, particularly affecting gray matter, has been attributed mainly to disturbances in the proliferation and survival of early neuronal progenitors. In addition, defects in dendritic development and synaptogenesis exist that affect the excitation-inhibition balance. Here, we studied proteomic changes in cerebral cortices of Cdk5rap2 mutant mice. Material and methods: : We used large-gel two-dimensional gel (2-DE) electrophoresis to separate cortical proteins. 2-DE gels were visualized by a trained observer on a light box. Spot changes were considered with respect to presence/absence, quantitative variation and altered mobility. Result: : We identified a reduction in more than 30 proteins that play a role in processes such as cell cytoskeleton dynamics, cell cycle progression, ciliary functions and apoptosis. These proteome changes in the MCPH3 model can be associated with various functional and morphological alterations of the developing brain. Conclusion: : Our results shed light on potential protein candidates for the disease-associated phenotype reported in MCPH3

Homozygous mutation in MCM7 causes autosomal recessive primary microcephaly and intellectual disability

Minichromosomal maintenance (MCM) complex components 2, 4, 5 and 6 have been linked to human disease with phenotypes including microcephaly and intellectual disability. The MCM complex has DNA helicase activity and is thereby important for the initiation and elongation of the replication fork and highly expressed in proliferating neural stem cells. Whole-exome sequencing was applied to identify the genetic cause underlying the neurodevelopmental disease of the index family. The expression pattern of was characterised by performing quantitative real-time PCR, hybridisation and immunostaining. To prove the disease-causative nature of identified , a proof-of-principle experiment was performed. We reported that the homozygous missense variant c.793G>A/p.A265T (g.7:99695841C>T, NM_005916.4) in was associated with autosomal recessive primary microcephaly (MCPH), severe intellectual disability and behavioural abnormalities in a consanguineous pedigree with three affected individuals. We found concordance between the spatiotemporal expression pattern of in mice and a proliferative state: expression was higher in early mouse developmental stages and in proliferative zones of the brain. Accordingly, Mcm7/MCM7 levels were detectable particularly in undifferentiated mouse embryonal stem cells and human induced pluripotent stem cells compared with differentiated neurons. We further demonstrate that the downregulation of in mouse neuroblastoma cells reduces cell viability and proliferation, and, as a proof-of-concept, that this is counterbalanced by the overexpression of wild-type but not mutant . We report mutations of as a novel cause of autosomal recessive MCPH and intellectual disability and highlight the crucial function of MCM7 in nervous system development

Identifizierung und Charakterisierung von Mechanismen assoziiert mit Intelligenzminderung

Intellectual disability (ID) has a high prevalence in individuals with neurodevelopmental disorders. Despite the high number of ID-associated genes, the genetic cause remains unclear in a considerable proportion of pedigrees and similarly the cellular and molecular mechanisms underlying the neurodevelopmental function of these genes remain poorly understood. The main aims of my PhD project was to (i) identify ‘novel’ genes causing ID and characterize their pathophysiologic role in brain development, and (ii) characterize pathomechanism of known ID disorders and expand their clinical and cellular phenotype. First, we approached the model disorder for congenital microcephaly and ID, autosomal recessive primary microcephaly (MCPH). MCPH is a clinically and genetically heterogeneous neurodevelopmental disorder with reduced brain volume at birth, ID, and lack of extracranial malformations. The hypothesis for MCPH-pathomechanism entails a premature shift from symmetric to asymmetric neuronal differentiation, resulting in progenitor pool depletion, thereby microcephaly and ID. Biallelic Cyclin-dependent kinase 5 regulatory- subunit associated protein 2 (CDK5RAP2) mutations cause MCPH3. Our data from Cdk5rap2-depleted murine embryonic stem cells revealed that in addition to premature neural differentiation, accumulating proliferating defect and increased apoptosis of differentiating- and early-postmitotic cells contribute to microcephaly. We reported for the first time the cellular phenotype of abnormal cell cycle apparatus (mitotic spindles, centrosomes), and lagging chromosomes in a MCPH2 patient with compound heterozygous WD-repeat domain 62 (WDR62) mutations. Reduced centrosomal CDK5RAP2 in MCPH2 patient’s cells indicates converging functional role of MCPH genes. Further, we identified a homozygous mutation in the previously not disease-linked gene Rho guanine nucleotide exchange factor 2 (ARHGEF2) in two patients with ID, congenital microcephaly, and mid-hindbrain malformation. We showed that the loss of ARHGEF2 causes abnormal mitotic spindles, spindle pole distance, impaired RhoA/ROCK/MLC pathway, and inhibits neurogenesis. We recapitulated the human brain phenotype in Arhgef2-/- mice and identified abnormal migration of precerebellar nuclei components as the underlying pathomechanism. Further, we reported a biallelic mutation in zinc-finger and BTB-domain containing protein 24 (ZBTB24) in a patient with immunodeficiency-centromeric instability-facial anomalies syndrome 2 (ICF2) characterized by immunodeficiency, developmental delay, and facial anomalies. Initially, ICF2 was acknowledged as an isolated B-cell defect. We extended the phenotype spectrum by describing the development of combined immunodeficiency with age in ICF2 as well as putative autoimmune phenomena (hepatitis, nephritis). We showed that impaired proliferation, increased apoptosis, and abnormal mitotic spindles are likely contribute to immunological and non-immunological phenotype in ICF2. With our studies, we demonstrated that abnormal proliferation, apoptosis, and/or migration due to defective cell cycle apparatus underlies as a common pathomechanism in ID and associated disorders.Eine Intelligenzminderung (ID) tritt mit einer hohen Prävalenz in Patienten mit einer neurologischen Entwicklungsstörung auf. Trotz der hohen Anzahl an ID-assoziierten Genen, bleibt die genetische Ursache sowie die zugrunde liegenden zellulären und molekularen Mechanismen in vielen Fällen noch unbekannt. Die Hauptziele meiner Promotionsarbeit waren (i) neue Gene zu identifizieren, die zu ID führen und deren pathogene Rolle in der Gehirnentwicklung zu untersuchen und (ii) den Pathomechanismus bekannter Erkrankungen mit ID zu charakterisieren und den klinischen und zellulären Phänotyp zu erweitern. In ersten Studien untersuchten wir die Modellerkrankung für angeborene Mikrozephalie und ID, die autosomal-rezessive primäre Mikrozephalie (MCPH). MCPH ist eine klinisch und genetisch heterogene Entwicklungsstörung des Gehirns, welche durch eine starke Verringerung des Hirnvolumens bei der Geburt, ID, sowie dem Fehlen von extrakraniellen Fehlbildungen gekennzeichnet ist. Die gegenwärtige Hypothese zur Entstehung von MCPH beschreibt eine frühzeitige Verschiebung der symmetrischen zu einer asymmetrischen neuronalen Proliferation, die zu einer Verringerung des Stammzellpools führt. Mutationen im Gen Cyclin-dependent kinase 5 regulatory- subunit associated protein 2 (CDK5RAP2) verursachen MCPH3. Unsere Experimente mit Cdk5rap2-herunterregulierten murinen embryonalen Stammzellen zeigten, dass neben einer vorzeitigen neuronalen Differenzierung, ebenfalls ein Proliferationsdefekt und eine erhöhte Apoptoserate von frühen postmitotischen Zellen zur Mikrozephalie beitragen. Zudem zeigt diese Studie zum ersten Mal Veränderungen im Aufbau des Zellzyklusapparates (Mitosespindel, Zentrosom) und der Chromosomenkondensierung in einem Patienten mit einer compound heterozygoten Mutation im WD-repeat domain 62 Gen (WDR62). In einer weiteren Studie wurde eine homozygote Mutation im bisher nicht krankheitsassoziierten Gen Rho guanine nucleotide exchange factor 2 (ARHGEF2) in zwei Patienten mit ID, kongenitaler Mikrozephalie und einer Fehlbildung des Mittel- und Hinterhirns untersucht. Wir konnten in funktionalen Studien zeigen, dass der Verlust von ARHGEF2 zu Veränderungen in Mitosespindeln und dem Spindelpolabstand sowie einer Beeinträchtigung des RhoA/ROCK/MLC-Signalweges führt, welches zur Hemmung der Neurogenese beiträgt. Der humane Phänotyp konnte in Arhgef2-knockout Mäusen rekapituliert werden und zeigte zudem eine gestörte Migration von präzerebellaren Zellen. Weiterhin berichten wir über eine biallelische Mutation im zinc-finger and BTB-domain containing protein 24 (ZBTB24) in einem Patienten mit immunodeficiency-centromeric instability- facial anomalies syndrome 2 (ICF2), charakterisiert durch Immunschwäche, Entwicklungsverzögerung und Gesichtsanomalien. Bislang wurde ICF2 primär als isolierter B-Zelldefekt anerkannt. Wir erweiterten das Phänotypspektrum, indem wir zum ersten Mal die Entwicklung einer kombinierten Immunschwäche und eines vermutlichen Autoimmunphänomens (Hepatitis, Nephritis) beschrieben. Weiterhin konnten wir nachweisen, dass Veränderungen in der Zellproliferation, Apoptoserate und Veränderung in der Chromosomentrennung zu dem beschriebenen immunologischen und nicht-immunologischen Phänotyp in ICF2 beitragen. Mit unseren Studien konnten wir nachweisen, dass eine abnormale Proliferation, Apoptose und / oder Migration aufgrund eines defekten Zellzyklusapparates als gemeinsamer Pathomechanismus bei ID und assoziierten Störungen zugrunde liegt

Congenital microcephaly-linked CDK5RAP2 affects eye development

Biallelic mutations in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause autosomal recessive primary microcephaly type 3 (MCPH3). MCPH is characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. Only recently, congenital cataracts were reported in four patients of one pedigree with MCPH3. Given the lack of a further pedigree with this phenotype, it remained unclear whether this was a true causal relationship. Here we support the link between CDK5RAP2 and eye development by showing that most Cdk5rap2 mutant mice (an/an) exhibit eye malformations ranging from reduced size of one or both eyes (microphthalmia) to total absence of both eyes (anophthalmia). We also detected increased apoptosis in the an/an retinal progenitor cells associated with more mitotic cells. This indicates an important role of Cdk5rap2 in physiologic eye development.This work was supported by the German Research Foundation (DFG, SFB665, SFB1315), the Helmholtz Association by the Berlin Institute of Health (BIH), the German Academic Exchange Service (DAAD), and the Charité – Universitätsmedizin Berlin

Case report: Compound heterozygous NUP85 variants cause autosomal recessive primary microcephaly

Nucleoporin (NUP) 85 is a member of the Y-complex of nuclear pore complex (NPC) that is key for nucleocytoplasmic transport function, regulation of mitosis, transcription, and chromatin organization. Mutations in various nucleoporin genes have been linked to several human diseases. Among them, NUP85 was linked to childhood-onset steroid-resistant nephrotic syndrome (SRNS) in four affected individuals with intellectual disability but no microcephaly. Recently, we broaden the phenotype spectrum of NUP85-associated disease by reporting NUP85 variants in two unrelated individuals with primary autosomal recessive microcephaly (MCPH) and Seckel syndrome (SCKS) spectrum disorders (MCPH-SCKS) without SRNS. In this study, we report compound heterozygous NUP85 variants in an index patient with only MCPH phenotype, but neither Seckel syndrome nor SRNS was reported. We showed that the identified missense variants cause reduced cell viability of patient-derived fibroblasts. Structural simulation analysis of double variants is predicted to alter the structure of NUP85 and its interactions with neighboring NUPs. Our study thereby further expands the phenotypic spectrum of NUP85-associated human disorder and emphasizes the crucial role of NUP85 in the brain development and function

Abnormal centrosome and spindle morphology in a patient with autosomal recessive primary microcephaly type 2 due to compound heterozygous WDR62 gene mutation

Background: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disease with severe microcephaly at birth due to a pronounced reduction in brain volume and intellectual disability. Biallelic mutations in the WD repeat-containing protein 62 gene WDR62 are the genetic cause of MCPH2. However, the exact underlying pathomechanism of MCPH2 remains to be clarified. Methods/results: We characterized the clinical, radiological, and cellular features that add to the human MCPH2 phenotype. Exome sequencing followed by Sanger sequencing in a German family with two affected daughters with primary microcephaly revealed in the index patient the compound heterozygous mutations c. 1313G>A (p.R438H) / c.2864-2867delACAG (p.D955Afs*112) of WDR62, the second of which is novel. Radiological examination displayed small frontal lobes, corpus callosum hypoplasia, simplified hippocampal gyration, and cerebellar hypoplasia. We investigated the cellular phenotype in patient-derived lymphoblastoid cells and compared it with that of healthy female controls. WDR62 expression in the patient's immortalized lymphocytes was deranged, and mitotic spindle defects as well as abnormal centrosomal protein localization were apparent. Conclusion: We propose that a disruption of centrosome integrity and/or spindle organization may play an important role in the development of microcephaly in MCPH2