ABSTRACT
Lamin B1 plays an important role in the nuclear envelope stability, the regulation of gene expression, and neural development. Duplication of LMNB1, or missense mutations increasing LMNB1 expression, are associated with autosomal-dominant leukodystrophy. On the basis of its role in neurogenesis, it has been postulated that LMNB1 variants could cause microcephaly. Here, we confirm this hypothesis with the identification of de novo mutations in LMNB1 in seven individuals with pronounced primary microcephaly (ranging from -3.6 to -12 SD) associated with relative short stature and variable degree of intellectual disability and neurological features as the core symptoms. Simplified gyral pattern of the cortex and abnormal corpus callosum were noted on MRI of three individuals, and these individuals also presented with a more severe phenotype. Functional analysis of the three missense mutations showed impaired formation of the LMNB1 nuclear lamina. The two variants located within the head group of LMNB1 result in a decrease in the nuclear localization of the protein and an increase in misshapen nuclei. We further demonstrate that another mutation, located in the coil region, leads to increased frequency of condensed nuclei and lower steady-state levels of lamin B1 in proband lymphoblasts. Our findings collectively indicate that de novo mutations in LMNB1 result in a dominant and damaging effect on nuclear envelope formation that correlates with microcephaly in humans. This adds LMNB1 to the growing list of genes implicated in severe autosomal-dominant microcephaly and broadens the phenotypic spectrum of the laminopathies.
Subject(s)
Dwarfism/genetics , Intellectual Disability/genetics , Lamin Type B/genetics , Microcephaly/genetics , Mutation , Nuclear Lamina/genetics , Amino Acid Sequence , Base Sequence , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Child, Preschool , Corpus Callosum/diagnostic imaging , Corpus Callosum/metabolism , Corpus Callosum/pathology , Dwarfism/diagnostic imaging , Dwarfism/metabolism , Dwarfism/pathology , Female , Gene Expression , Humans , Infant , Intellectual Disability/diagnostic imaging , Intellectual Disability/metabolism , Intellectual Disability/pathology , Lamin Type B/metabolism , Lymphocytes/metabolism , Lymphocytes/pathology , Magnetic Resonance Imaging , Male , Microcephaly/diagnostic imaging , Microcephaly/metabolism , Microcephaly/pathology , Nuclear Lamina/metabolism , Nuclear Lamina/pathologyABSTRACT
BACKGROUND: C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. In humans, biallelic variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux, while heterozygous variants in NPR2 (natriuretic peptide receptor 2) and NPPC (natriuretic peptide precursor C), encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the protein kinase cGMP-dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been clearly demonstrated in several animal models. METHODS: Exome sequencing was performed in two girls with severe short stature due to acromesomelic limb shortening, brachydactyly, mild to moderate platyspondyly and progressively increasing metaphyseal alterations of the long bones. Functional characterisation was undertaken for the identified variants. RESULTS: Two homozygous PRKG2 variants, a nonsense and a frameshift, were identified. The mutant transcripts are exposed to nonsense-mediated decay and the truncated mutant cGKII proteins, partially or completely lacking the kinase domain, alter the downstream mitogen activation protein kinase signalling pathway by failing to phosphorylate c-Raf 1 at Ser43 and subsequently reduce ERK1/2 activation in response to fibroblast growth factor 2. They also downregulate COL10A1 and upregulate COL2A1 expression through SOX9. CONCLUSION: In conclusion, we have clinically and molecularly characterised a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP).
Subject(s)
Cyclic GMP-Dependent Protein Kinase Type II/genetics , Dwarfism/genetics , Mutation , Osteochondrodysplasias/genetics , Brachydactyly , Child , Dwarfism/metabolism , Female , Humans , Osteochondrodysplasias/metabolism , Pedigree , Exome SequencingABSTRACT
WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.
Subject(s)
Cell Cycle Proteins/metabolism , Cilia/metabolism , Ciliopathies/genetics , Microcephaly/genetics , Microtubule-Associated Proteins/metabolism , Neocortex/metabolism , Nerve Tissue Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Anophthalmos/embryology , Anophthalmos/genetics , Anophthalmos/metabolism , Apoptosis/genetics , CRISPR-Cas Systems , Cell Cycle Proteins/genetics , Cells, Cultured , Cilia/genetics , Cilia/pathology , Ciliopathies/embryology , Ciliopathies/metabolism , Ciliopathies/pathology , Dwarfism/embryology , Dwarfism/genetics , Dwarfism/metabolism , Ependymoglial Cells/cytology , Ependymoglial Cells/metabolism , Ependymoglial Cells/pathology , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microcephaly/embryology , Microcephaly/metabolism , Microtubule-Associated Proteins/genetics , Mutation, Missense , Neocortex/embryology , Nerve Tissue Proteins/genetics , Neurogenesis/genetics , Neuroglia/cytology , Neuroglia/metabolism , Neurons/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.
Subject(s)
Dwarfism , Osteogenesis , Animals , Cell Differentiation , Cell Proliferation , Core Binding Factor Alpha 1 Subunit/metabolism , Dwarfism/genetics , Dwarfism/metabolism , Hedgehog Proteins/metabolism , Mice , Osteoblasts/metabolism , Osteogenesis/physiology , Phenotype , Sp7 Transcription Factor/metabolism , Transcription Factors/geneticsABSTRACT
Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up-regulated circANAPC2 was identified by circRNA microarray analysis and RT-qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down-regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR-874-3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR-874-3p. The co-localization of circANAPC2 and miR-874-3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR-874-3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high-throughput screening of mRNA expression and RT-qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR-874-3p could be targeted by Smad3, thus down-regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR-874-3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.
Subject(s)
Chondrocytes/metabolism , Dwarfism/genetics , Dwarfism/metabolism , MicroRNAs/metabolism , Osteogenesis , RNA, Circular/metabolism , Smad3 Protein/metabolism , Body Height , Cell Proliferation , Child , Child, Preschool , Down-Regulation , Female , High-Throughput Nucleotide Sequencing , Humans , Male , MicroRNAs/genetics , RNA, Circular/genetics , Signal Transduction , Smad3 Protein/genetics , Up-RegulationABSTRACT
Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.
Subject(s)
Chondrocytes/cytology , Chondrogenesis , Craniofacial Abnormalities/metabolism , Dwarfism/metabolism , Extremities/embryology , Limb Deformities, Congenital/metabolism , Urogenital Abnormalities/metabolism , Wnt-5a Protein/genetics , Animals , Animals, Genetically Modified , Cartilage/metabolism , Cell Polarity/physiology , Chick Embryo , Chickens , Chondrogenesis/genetics , Craniofacial Abnormalities/genetics , Disease Models, Animal , Dwarfism/genetics , HEK293 Cells , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Limb Deformities, Congenital/genetics , Mutation, Missense , Phenotype , Urogenital Abnormalities/genetics , Wnt Signaling Pathway , Wnt-5a Protein/metabolismABSTRACT
Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.
Subject(s)
Craniofacial Abnormalities/veterinary , Dishevelled Proteins/genetics , Dog Diseases/genetics , Dogs/genetics , Dwarfism/veterinary , Limb Deformities, Congenital/veterinary , Urogenital Abnormalities/veterinary , Amino Acid Sequence , Animals , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Dishevelled Proteins/metabolism , Dog Diseases/metabolism , Dogs/anatomy & histology , Dogs/classification , Dwarfism/genetics , Dwarfism/metabolism , Female , Frameshift Mutation , Genetic Variation , Genome-Wide Association Study , Humans , Limb Deformities, Congenital/genetics , Limb Deformities, Congenital/metabolism , Male , Organosilicon Compounds , Sequence Homology, Amino Acid , Species Specificity , Tail/anatomy & histology , Urogenital Abnormalities/genetics , Urogenital Abnormalities/metabolism , Wnt Signaling Pathway/geneticsABSTRACT
Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-ß in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.
Subject(s)
Bone Diseases/congenital , Dwarfism/metabolism , Osteoblasts/pathology , Proteoglycans/metabolism , Skin Diseases, Genetic/metabolism , Transforming Growth Factor beta/metabolism , Vesicular Transport Proteins/metabolism , Animals , Bone Diseases/metabolism , Bone Diseases/pathology , Cell Differentiation , Decorin/metabolism , Dermatan Sulfate/metabolism , Disease Models, Animal , Dwarfism/pathology , Female , Fractures, Bone/genetics , Glycosylation , Golgi Matrix Proteins , Mesenchymal Stem Cells/pathology , Mesenchymal Stem Cells/physiology , Mice, Inbred C57BL , Mice, Transgenic , Osteoblasts/metabolism , Signal Transduction , Skin Diseases, Genetic/pathology , Vesicular Transport Proteins/geneticsABSTRACT
The complexity of skeletal pathologies makes use of in vivo models essential to elucidate the pathogenesis of the diseases; nevertheless, chondrocyte and osteoblast cell lines provide relevant information on the underlying disease mechanisms. Due to the limitations of primary chondrocytes, immortalized cells represent a unique tool to overcome this problem since they grow very easily for several passages. However, in the immortalization procedure the cells might lose the original phenotype; thus, these cell lines should be deeply characterized before their use. We immortalized primary chondrocytes from a Cant1 knock-out mouse, an animal model of Desbuquois dysplasia type 1, with a plasmid expressing the SV40 large and small T antigen. This cell line, based on morphological and biochemical parameters, showed preservation of the chondrocyte phenotype. In addition reduced proteoglycan synthesis and oversulfation of glycosaminoglycan chains were demonstrated, as already observed in primary chondrocytes from the Cant1 knock-out mouse. In conclusion, immortalized Cant1 knock-out chondrocytes maintained the disease phenotype observed in primary cells validating the in vitro model and providing an additional tool to further study the proteoglycan biosynthesis defect. The same approach might be extended to other cartilage disorders.
Subject(s)
Acid Anhydride Hydrolases/physiology , Chondrocytes/pathology , Craniofacial Abnormalities/pathology , Dwarfism/pathology , Glycosaminoglycans/metabolism , Joint Instability/pathology , Ossification, Heterotopic/pathology , Phenotype , Polydactyly/pathology , Animals , Cell Line, Transformed , Chondrocytes/metabolism , Craniofacial Abnormalities/etiology , Craniofacial Abnormalities/metabolism , Dwarfism/etiology , Dwarfism/metabolism , Joint Instability/etiology , Joint Instability/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Ossification, Heterotopic/etiology , Ossification, Heterotopic/metabolism , Polydactyly/etiology , Polydactyly/metabolismABSTRACT
Calcineurin is a calcium (Ca2+)/calmodulin-regulated protein phosphatase that mediates Ca2+-dependent signal transduction. Here, we report six heterozygous mutations in a gene encoding the alpha isoform of the calcineurin catalytic subunit (PPP3CA). Notably, mutations were observed in different functional domains: in addition to three catalytic domain mutations, two missense mutations were found in the auto-inhibitory (AI) domain. One additional frameshift insertion that caused premature termination was also identified. Detailed clinical evaluation of the six individuals revealed clinically unexpected consequences of the PPP3CA mutations. First, the catalytic domain mutations and frameshift mutation were consistently found in patients with nonsyndromic early onset epileptic encephalopathy. In contrast, the AI domain mutations were associated with multiple congenital abnormalities including craniofacial dysmorphism, arthrogryposis and short stature. In addition, one individual showed severe skeletal developmental defects, namely, severe craniosynostosis and gracile bones (severe bone slenderness and perinatal fractures). Using a yeast model system, we showed that the catalytic and AI domain mutations visibly result in decreased and increased calcineurin signaling, respectively. These findings indicate that different functional effects of PPP3CA mutations are associated with two distinct disorders and suggest that functional approaches using a simple cellular system provide a tool for resolving complex genotype-phenotype correlations.
Subject(s)
Arthrogryposis/genetics , Calcineurin/genetics , Craniofacial Abnormalities/genetics , Dwarfism/genetics , Gain of Function Mutation , Loss of Function Mutation , Spasms, Infantile/genetics , Amino Acid Sequence , Arthrogryposis/metabolism , Arthrogryposis/pathology , Base Sequence , Calcineurin/chemistry , Calcineurin/metabolism , Child , Child, Preschool , Craniofacial Abnormalities/metabolism , Craniofacial Abnormalities/pathology , Dwarfism/metabolism , Dwarfism/pathology , Female , Gene Expression Regulation , Genetic Association Studies , Humans , Male , Models, Molecular , Pedigree , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spasms, Infantile/metabolism , Spasms, Infantile/pathology , Young AdultABSTRACT
Mutations in the a disintegrin and metalloproteinase with thrombospondin motif-like 2 ( ADAMTSL2) gene are responsible for the autosomal recessive form of geleophysic dysplasia, which is characterized by short stature, short extremities, and skeletal abnormalities. However, the exact function of ADAMTSL2 is unknown. To elucidate the role of this protein in skeletal development, we generated complementary knockout (KO) mouse models with either total or chondrocyte Adamtsl2 deficiency. We observed that the Adamtsl2 KO mice displayed skeletal abnormalities reminiscent of the human phenotype. Adamtsl2 deletion affected the growth plate formation with abnormal differentiation and proliferation of chondrocytes. In addition, a TGF-ß signaling impairment in limbs lacking Adamtsl2 was demonstrated. Further investigations revealed that Adamtsl2 KO chondrocytes failed to establish a microfibrillar network composed by fibrillin1 and latent TGF-ß binding protein 1 fibrils. Chondrocyte Adamtsl2 KO mice also exhibited dwarfism. These studies uncover the function of Adamtsl2 in the maintenance of the growth plate ECM by modulating the microfibrillar network.-Delhon, L., Mahaut, C., Goudin, N., Gaudas, E., Piquand, K., Le Goff, W., Cormier-Daire, V., Le Goff, C. Impairment of chondrogenesis and microfibrillar network in Adamtsl2 deficiency.
Subject(s)
ADAMTS Proteins/physiology , Bone Diseases, Developmental/etiology , Chondrogenesis , Dwarfism/etiology , Extracellular Matrix Proteins/physiology , Microfibrils/pathology , Animals , Bone Diseases, Developmental/metabolism , Bone Diseases, Developmental/pathology , Dwarfism/metabolism , Dwarfism/pathology , Heterozygote , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfibrils/metabolism , Mutation , Phenotype , Transforming Growth Factor beta/metabolismABSTRACT
The RING finger protein TRAIP protects genome integrity and its mutation causes Seckel syndrome. TRAIP encodes a nucleolar protein that migrates to UV-induced DNA lesions via a direct interaction with the DNA replication clamp PCNA. Thus far, mechanistically how UV mobilizes TRAIP from the nucleoli remains unknown. We found that PCNA binding is dispensable for the nucleolus-nucleoplasm shuttling of TRAIP following cell exposure to UV irradiation, and that its redistribution did not rely on the master DNA damage kinases ATM and ATR. Interestingly, I-PpoI-induced ribosomal DNA damage led to TRAIP exclusion from the nucleoli, raising the possibility that active ribosomal DNA transcription may underlie TRAIP retention in the nuclear sub-compartments. Accordingly, chemical inhibition of RNA polymerase I activity led to TRAIP diffusion into the nucleoplasm, and was coupled with marked reduction of DNA/RNA hybrids in the nucleoli, suggesting that TRAIP may be sequestered via binding to nucleic acid structures in the nucleoli. Consistently, cell pre-treatment with DNase/RNase effectively released TRAIP from the nucleoli. Taken together, our study defines a bipartite mechanism that drives TRAIP trafficking in response to UV damage, and highlights the nucleolus as a stress sensor that contributes to orchestrating DNA damage responses.
Subject(s)
Cell Nucleolus/metabolism , DNA, Ribosomal/genetics , RNA Polymerase I/genetics , Transcription, Genetic , Ubiquitin-Protein Ligases/genetics , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Benzothiazoles/pharmacology , Cell Line, Tumor , Cell Nucleolus/radiation effects , Cell Nucleolus/ultrastructure , DNA Damage , DNA, Ribosomal/metabolism , Deoxyribonucleases/chemistry , Dwarfism/genetics , Dwarfism/metabolism , Dwarfism/pathology , Facies , Gene Expression Regulation , HeLa Cells , Humans , Microcephaly/genetics , Microcephaly/metabolism , Microcephaly/pathology , Naphthyridines/pharmacology , Osteoblasts/metabolism , Osteoblasts/pathology , Osteoblasts/radiation effects , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Transport , RNA Polymerase I/antagonists & inhibitors , RNA Polymerase I/metabolism , Ribonucleases/chemistry , Ribosomes/genetics , Ribosomes/metabolism , Ubiquitin-Protein Ligases/metabolism , Ultraviolet RaysABSTRACT
To ensure that the genetic material is accurately passed down to daughter cells during mitosis, dividing cells must duplicate their chromosomes and centrosomes once and only once per cell cycle. The same key steps-licensing, duplication, and segregation-control both the chromosome and the centrosome cycle, which must occur in concert to safeguard genome integrity. Aberrations in genome content or centrosome numbers lead to genomic instability and are linked to tumorigenesis. Such aberrations, however, can also be part of the normal life cycle of specific cell types. Multiciliated cells best exemplify the deviation from a normal centrosome cycle. They are post-mitotic cells which massively amplify their centrioles, bypassing the rule for once-per-cell-cycle centriole duplication. Hundreds of centrioles dock to the apical cell surface and generate motile cilia, whose concerted movement ensures fluid flow across epithelia. The early steps that control the generation of multiciliated cells have lately started to be elucidated. Geminin and the vertebrate-specific GemC1 and McIdas are distantly related coiled-coil proteins, initially identified as cell cycle regulators associated with the chromosome cycle. Geminin is required to ensure once-per-cell-cycle genome replication, while McIdas and GemC1 bind to Geminin and are implicated in DNA replication control. Recent findings highlight Geminin family members as early regulators of multiciliogenesis. GemC1 and McIdas specify the multiciliate cell fate by forming complexes with the E2F4/5 transcription factors to switch on a gene expression program leading to centriole amplification and cilia formation. Positive and negative interactions among Geminin family members may link cell cycle control to centriole amplification and multiciliogenesis, acting close to the point of transition from proliferation to differentiation. We review key steps of centrosome duplication and amplification, present the role of Geminin family members in the centrosome and chromosome cycle, and discuss links with disease.
Subject(s)
Centrioles/metabolism , Cilia/metabolism , Geminin/genetics , Genome , Mitosis , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinogenesis/pathology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Centrioles/ultrastructure , Cilia/ultrastructure , DNA Replication , Dwarfism/genetics , Dwarfism/metabolism , Dwarfism/pathology , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , E2F5 Transcription Factor/genetics , E2F5 Transcription Factor/metabolism , Geminin/metabolism , Gene Expression Regulation , Genomic Instability , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Signal Transduction , Transcription FactorsABSTRACT
Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We identified a syndrome due to an autosomal-recessively inherited deficiency of transketolase, encoded by TKT, on chromosome 3p21. Our series includes three families with a total of five affected individuals, ranging in age from 4 to 25 years. Two families of Ashkenazi Jewish ancestry were homozygous for an 18 base pair in-frame insertion in TKT. The third family was compound heterozygous for nonsense and missense variants in TKT. All affected individuals had short stature and were developmentally delayed. Congenital heart defects were noted in four of the five affected individuals, and there was a history of chronic diarrhea and cataracts in the older individuals with the homozygous 18 base pair insertion. Enzymatic testing confirmed significantly reduced transketolase activity. Elevated urinary excretion of erythritol, arabitol, ribitol, and pent(ul)ose-5-phosphates was detected, as well as elevated amounts of erythritol, arabitol, and ribitol in the plasma of affected individuals. Transketolase deficiency reduces NADPH synthesis and nucleic acid synthesis and cell division and could explain the problems with growth. NADPH is also critical for maintaining cerebral glutathione, which might contribute to the neurodevelopmental delays. Transketolase deficiency is one of a growing list of inborn errors of metabolism in the non-oxidative part of the pentose phosphate pathway.
Subject(s)
Developmental Disabilities/etiology , Dwarfism/etiology , Heart Defects, Congenital/etiology , Mutation/genetics , Transketolase/genetics , Adult , Child , Child, Preschool , Developmental Disabilities/metabolism , Developmental Disabilities/pathology , Dwarfism/metabolism , Dwarfism/pathology , Female , Glutathione/metabolism , Heart Defects, Congenital/metabolism , Heart Defects, Congenital/pathology , Humans , Male , NADP/metabolism , Pedigree , Syndrome , Young AdultABSTRACT
3M syndrome is characterized by severe pre- and postnatal growth retardation, typical facial features, and normal intelligence. Homozygous or compound heterozygous mutations in either CUL7, OBSL1, or CCDC8 have been identified in the etiology so far. Clinical and molecular features of 24 patients (23 patients and a fetus) from 19 unrelated families with a clinical diagnosis of 3M syndrome were evaluated and genotype-phenotype correlations were investigated with the use of DNA sequencing, chromosomal microarray, and whole exome sequencing accordingly. A genetic etiology could be established in 20 patients (n = 20/24, 83%). Eleven distinct CUL7 or OBSL1 mutations, among which eight was novel, were identified in 18 patients (n = 18/24, 75%). Ten patients had CUL7 (n = 10/18, 56%) while eight had OBSL1 (n = 8/18, 44%) mutations. Birth weight and height standard deviation scores at admission were significantly (p < 0.05) lower in patients with CUL7 mutation compared to that of patients with OBSL1 mutation. Two patients with a similar phenotype had a de novo 20p13p deletion involving BMP2. No genetic etiology could be established in four patients (n = 4/28, 17%). This study yet represents the largest cohort of 3M syndrome patients from a single center in Turkey. Microdeletions involving BMP2 may cause a phenotype similar to 3M syndrome with some distinctive features. Larger cohort of patients are required to establish genotype-phenotype correlations in 3M syndrome.
Subject(s)
Bone Morphogenetic Protein 2/genetics , Cullin Proteins/genetics , Cytoskeletal Proteins/genetics , Dwarfism/genetics , Genetic Association Studies , Muscle Hypotonia/genetics , Mutation , Spine/abnormalities , Adolescent , Base Sequence , Bone Morphogenetic Protein 2/deficiency , Child , Child, Preschool , Chromosomes, Human, Pair 20 , Cohort Studies , Cullin Proteins/metabolism , Cytoskeletal Proteins/metabolism , Dwarfism/diagnosis , Dwarfism/metabolism , Dwarfism/pathology , Female , Fetus , Gene Expression , Genotype , Humans , Infant , Infant, Newborn , Male , Muscle Hypotonia/diagnosis , Muscle Hypotonia/metabolism , Muscle Hypotonia/pathology , Phenotype , Spine/metabolism , Spine/pathology , Exome SequencingABSTRACT
AIM: To determine whether non-obese prepubertal children with growth hormone deficiency (GHD) present changes in lipid metabolism, and adipokines, and to assess the short-term effects of growth hormone (GH) treatment on these parameters. METHODS: Prospective observational follow-up and case-control (36 GHD children and 38 healthy children) study lasted for six months. Means of values from groups were compared, control group versus GHD baseline group, and GHD baseline group versus GHD after six months of GH replacement therapy. Lipid profile, glucose, insulin, homeostatic model assessment - insulin resistance (HOMA-IR), leptin, adiponectin and soluble intercellular adhesion molecule-1 (sICAM-1) were all analysed. RESULTS: Growth hormone deficiency children show higher baseline levels of total cholesterol, LDL cholesterol, triglycerides, Apo B and sICAM-1, but lower levels of free fatty acids, insulin and HOMA-IR. After six months of treatment, cholesterol, LDL cholesterol, Apo B, T cholesterol/HDL cholesterol, insulin, HOMA-IR and leptin levels decreased. The changes in insulin and HOMA-IR levels correlated inversely with the changes in HDL cholesterol and Apo A1 levels. A correlation was also observed between the changes in adiponectin levels and the changes in HDL cholesterol and Apo A1 levels. Variations in leptin levels were correlated with changes in triglycerides. CONCLUSION: Prepubertal non-obese GHD children present altered lipid profiles and adipokine levels. Replacement therapy with GH improves these variables.
Subject(s)
Adipokines/metabolism , Dwarfism/drug therapy , Dwarfism/metabolism , Endothelium, Vascular/physiopathology , Hormone Replacement Therapy , Human Growth Hormone/deficiency , Human Growth Hormone/therapeutic use , Lipid Metabolism , Case-Control Studies , Child , Endothelium, Vascular/drug effects , Female , Follow-Up Studies , Human Growth Hormone/pharmacology , Humans , Lipid Metabolism/drug effects , Male , Prospective StudiesABSTRACT
WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.
Subject(s)
Amino Acid Transport Systems, Acidic/deficiency , Antiporters/deficiency , Cerebral Cortex/metabolism , Dwarfism/genetics , Epilepsy/genetics , Hereditary Central Nervous System Demyelinating Diseases/genetics , Mitochondrial Diseases/genetics , Neurogenesis/genetics , Psychomotor Disorders/genetics , Tumor Suppressor Proteins/genetics , WW Domain-Containing Oxidoreductase/genetics , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase/genetics , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase/metabolism , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/metabolism , Amino Acid Transport Systems, Acidic/genetics , Amino Acid Transport Systems, Acidic/metabolism , Animals , Antiporters/genetics , Antiporters/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Cell Count , Cerebral Cortex/growth & development , Cerebral Cortex/pathology , Disease Models, Animal , Dwarfism/metabolism , Dwarfism/pathology , Epilepsy/metabolism , Epilepsy/pathology , Gene Expression Regulation, Developmental , Germ-Line Mutation , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Hereditary Central Nervous System Demyelinating Diseases/metabolism , Hereditary Central Nervous System Demyelinating Diseases/pathology , Male , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Myelin Basic Protein/genetics , Myelin Basic Protein/metabolism , Neurons/metabolism , Neurons/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , Prosencephalon/growth & development , Prosencephalon/metabolism , Prosencephalon/pathology , Psychomotor Disorders/metabolism , Psychomotor Disorders/pathology , Rats , Rats, Transgenic , Signal Transduction , Tumor Suppressor Proteins/deficiency , WW Domain-Containing Oxidoreductase/deficiencyABSTRACT
Ataxia Telangiectasia and Rad3 related (ATR) is one of the main regulators of the DNA damage response. It coordinates cell cycle checkpoint activation, replication fork stability, restart and origin firing to maintain genome integrity. Mutations of the ATR gene have been reported in Seckel patients, who suffer from a rare genetic disease characterized by severe microcephaly and growth retardation. Here, we report the case of a Seckel patient with compound heterozygous mutations in ATR. One allele has an intronic mutation affecting splicing of neighboring exons, the other an exonic missense mutation, producing the variant p.Lys1665Asn, of unknown pathogenicity. We have modeled this novel missense mutation, as well as a previously described missense mutation p.Met1159Ile, and assessed their effect on ATR function. Interestingly, our data indicate that both missense mutations have no direct effect on protein function, but rather result in defective ATR splicing. These results emphasize the importance of splicing mutations in Seckel Syndrome.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Dwarfism/genetics , Microcephaly/genetics , Mutation, Missense , RNA Splicing , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line , Chickens , Dwarfism/metabolism , Exons , Humans , Introns , Microcephaly/metabolism , Exome SequencingABSTRACT
Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS.
Subject(s)
Congenital Microtia/genetics , Dwarfism/genetics , Geminin/genetics , Growth Disorders/genetics , Micrognathism/genetics , Mutation , Patella/abnormalities , Adolescent , Amino Acid Sequence , Base Sequence , Cell Cycle/genetics , Child, Preschool , Congenital Microtia/metabolism , Dwarfism/metabolism , Dwarfism/pathology , Exons , Female , Geminin/metabolism , Gene Expression , Genes, Dominant , Growth Disorders/metabolism , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Inheritance Patterns , Male , Micrognathism/metabolism , Molecular Sequence Data , Patella/metabolism , Pedigree , Protein Stability , Proteolysis , RNA Splicing , Sequence AlignmentABSTRACT
The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.