RESUMO
BACKGROUND: Complex regional pain syndrome type 1 (CRPS-1) is a rare, disabling and sometimes chronic disorder usually arising after a trauma. This exploratory study examined whether patients with chronic CRPS-1 have a different genetic profile compared with those who do not have the condition. METHODS: Exome sequencing was performed to seek altered non-synonymous SNP allele frequencies in a discovery cohort of well-characterised patients with chronic CRPS-1 (n=34) compared with population databases. Identified SNP alleles were confirmed by Sanger sequencing and sought in a replication cohort (n=50). Gene expression of peripheral blood macrophages was assessed. RESULTS: In the discovery cohort, the rare allele frequencies of four non-synonymous SNPs were statistically increased. The replication cohort confirmed this finding. In a chronic pain cohort, these alleles were not overexpressed. In total, 25 out of 84 (29.8%) patients with CRPS-1 expressed a rare allele. The SNPs were rs41289586 in ANO10, rs28360457 in P2RX7, rs1126930 in PRKAG1 and rs80308281 in SLC12A9. Males were more likely than females to have a rare SNP allele, 8 out of 14 (57.1%) vs 17 out of 70 (24.3%) (Fisher's p=0.023). ANO10, P2RX7, PRKAG1 and SLC12A9 were all expressed in macrophages from healthy human controls. CONCLUSION: A single SNP in each of the genes ANO10, P2RX7, PRKAG1 and SLC12A9 was associated with developing chronic CRPS-1, with more males than females expressing these rare alleles. Our work suggests the possibility that a permissive genetic background is an important factor in the development of CRPS-1.
Assuntos
Síndromes da Dor Regional Complexa , Masculino , Feminino , Humanos , Síndromes da Dor Regional Complexa/genética , Síndromes da Dor Regional Complexa/epidemiologia , Frequência do Gene , Polimorfismo de Nucleotídeo Único/genética , Alelos , Patrimônio GenéticoRESUMO
BACKGROUND: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. METHODS: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. RESULTS: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. CONCLUSION: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.
Assuntos
Holoprosencefalia , Fosfolipases Tipo C , Animais , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Holoprosencefalia/metabolismo , Humanos , Mamíferos/metabolismo , Mutação , Fenótipo , Fosfolipases Tipo C/genéticaRESUMO
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Assuntos
Epilepsia/genética , Proteínas/genética , Espasmos Infantis/genética , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantis/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes.
Assuntos
Encefalopatias/genética , Proteínas/genética , Enzimas de Conjugação de Ubiquitina/genética , Adolescente , Adulto , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encefalopatias/fisiopatologia , Criança , Pré-Escolar , Feminino , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Mutação , Linhagem , Processamento de Proteína Pós-Traducional , Proteínas/fisiologia , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/fisiologiaRESUMO
BACKGROUND: Progressive encephalopathy, hypsarrhythmia and optic atrophy (PEHO) has been described as a clinically distinct syndrome. It has been postulated that it is an autosomal recessive condition. However, the aetiology is poorly understood, and the genetic basis of the condition has not been fully elucidated. Our objective was to discover if PEHO syndrome is a single gene disorder. METHOD: Children with PEHO and PEHO-like syndrome were recruited. Clinical, neurological and dysmorphic features were recorded; EEG reports and MRI scans were reviewed. Where possible, exome sequencing was carried out first to seek mutations in known early infantile developmental and epileptic encephalopathy (DEE) genes and then to use an agnostic approach to seek novel candidate genes. We sought intra-interfamilial phenotypic correlations and genotype-phenotype correlations when pathological mutations were identified. RESULTS: Twenty-three children were recruited from a diverse ethnic background, 19 of which were suitable for inclusion. They were similar in many of the core and the supporting features of PEHO, but there was significant variation in MRI and ophthalmological findings, even between siblings with the same mutation. A pathogenic genetic variant was identified in 15 of the 19 children. One further girl's DNA failed analysis, but her two affected sisters shared confirmed variants. Pathogenic variants were identified in seven different genes. CONCLUSIONS: We found significant clinical and genetic heterogeneity. Given the intrafamily variation demonstrated, we question whether the diagnostic criteria for MRI and ophthalmic findings should be altered. We also question whether PEHO and PEHO-like syndrome represent differing points on a clinical spectrum of the DEE. We conclude that PEHO and PEHO-like syndrome are clinically and genetically diverse entities-and are phenotypic endpoints of many severe genetic encephalopathies.
Assuntos
Edema Encefálico/diagnóstico , Edema Encefálico/etiologia , Epilepsia/diagnóstico , Epilepsia/genética , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/etiologia , Atrofia Óptica/diagnóstico , Atrofia Óptica/etiologia , Espasmos Infantis/diagnóstico , Espasmos Infantis/etiologia , Fatores Etários , Alelos , Biomarcadores , Pré-Escolar , Eletroencefalografia , Fácies , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Genótipo , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Mutação , Linhagem , FenótipoRESUMO
Bi-allelic dysfunctional mutations in nerve growth factor (NGF) cause the rare human phenotype hereditary sensory and autonomic neuropathy type 5 (HSAN5). We describe a novel NGF mutation in an individual with typical HSAN5 findings. The mutation c.361C>T, p.R121W is at the last residue of the furin cleavage motif Arg-Ser-Lys-Arg in proNGF. We show that the p.R121W mutation completely abolishes the formation of mature NGF-ß. Surprisingly, mutant p.R121W cells produced very little proNGF. Instead, the two progressive cleavage products of proNGF were produced, proA-NGF and proB-NGF, with proB-NGF being the predominant NGF-derived peptide and the only peptide secreted by mutant p.R121W cells. We found that the ability of the p.R121W mutation to cause tropomyosin receptor kinase A autophosphorylation and mitogen-activated protein kinase phosphorylation was significantly reduced compared to controls (p < 0.05 and p < 0.01). By studying the PC12 cell line morphology and neurite length over a week, we found the p.R121W mutation had residual, but much reduced, neurotrophic activity when compared to wild-type NGF. Finally, we assessed whether the p.R121W mutation affected apoptosis and found a reduced protective effect compared to wild-type NGF. Our results suggest that the p.R121W NGF mutation causes HSAN5 through negating the ability of furin to cleave proNGF to produce NGF-ß.
Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Mutação/genética , Fator de Crescimento Neural/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Animais , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Células PC12 , Fosforilação/genética , Precursores de Proteínas/metabolismo , RatosRESUMO
Hereditary sensory and autonomic neuropathy type IV (HSAN IV) is an autosomal recessive disorder characterized by a complete lack of pain perception and anhidrosis. Here, we studied a cohort of seven patients with HSAN IV and describe a comprehensive functional analysis of seven novel NTRK1 missense mutations, c.1550G >A, c.1565G >A, c.1970T >C, c.2096T >C, c.2254T >A, c.2288G >C, and c.2311C >T, corresponding to p.G517E, p.G522E, p.L657P, p.I699T, p.C752S, p.C763S, and p.R771C, all of which were predicted pathogenic by in silico analysis. The results allowed us to assess the pathogenicity of each mutation and to gain novel insights into tropomyosin receptor kinase A (TRKA) downstream signaling. Each mutation was systematically analyzed for TRKA glycosylation states, intracellular and cell membrane expression patterns, nerve growth factor stimulated TRKA autophosphorylation, TRKA-Y496 phosphorylation, PLCγ activity, and neurite outgrowth. We showed a diverse range of functional effects: one mutation appeared fully functional, another had partial activity in all assays, one mutation affected only the PLCγ pathway and four mutations were proved null in all assays. Thus, we conclude that complete abolition of TRKA kinase activity is not the only pathogenic mechanism underlying HSAN IV. By corollary, the assessment of the clinical pathogenicity of HSAN IV mutations is more complex than initially predicted and requires a multifaceted approach.
Assuntos
Neuropatias Hereditárias Sensoriais e Autônomas/genética , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Mutação de Sentido Incorreto , Receptor trkA/genética , Receptor trkA/metabolismo , Alelos , Linhagem Celular , Biologia Computacional/métodos , Análise Mutacional de DNA , Ordem dos Genes , Estudos de Associação Genética , Loci Gênicos , Predisposição Genética para Doença , Genótipo , Glicosilação , Neuropatias Hereditárias Sensoriais e Autônomas/diagnóstico , Humanos , Imagem Molecular , Neuritos/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptor trkA/química , Proteínas Recombinantes de Fusão , Análise de Sequência de DNA , Transdução de SinaisRESUMO
BACKGROUND: Significant human diseases/phenotypes exist which require both an environmental trigger event and a genetic predisposition before the disease/phenotype emerges, e.g. Carbamazepine with the rare SNP allele of rs3909184 causing Stevens Johnson syndrome, and aminoglycosides with rs267606617 causing sensory neural deafness. The underlying genotypes are fully penetrant only when the correct environmental trigger(s) occur, otherwise they are silent and harmless. Such diseases/phenotypes will not appear to have a Mendelian inheritance pattern, unless the environmental trigger is very common (>50% per lifetime). The known causative genotypes are likely to be protein-altering SNPs with dominant/semi-dominant effect. We questioned whether other diseases and phenotypes could have a similar aetiology. METHODS: We wrote the fSNPd program to analyse multiple exomes from a test cohort simultaneously with the purpose of identifying SNP alleles at a significantly different frequency to that of the general population. fSNPd was tested on trial cohorts, iteratively improved, and modelled for performance against an idealised association study under mutliple parameters. We also assessed the seqeuncing depath of all human exons to determine which were sufficiently well sequenced in an exome to be sued by fSNPd - by assessing forty exomes base by base. RESULTS: We describe a simple methodology for the detection of SNPs capable of causing a phenotype triggered by an environmental event. This uses cohorts of relatively small size (30-100 individuals) with the phenotype being investigated, their exomes, and thence seeks SNP allele frequencies significantly different from expected to identify potentially clinically important, protein altering SNP alleles. The strengths and weaknesses of this approach for discovering significant genetic causes of human disease are comparable to Mendelian disease mutation detection and Association Studies. CONCLUSIONS: The fSNPd methodology is another approach, and has potentially significant advantage over Association studies in needing far fewer individuals, to detect genes involved in the pathogenesis of a diseases/phenotypes. Furthermore, the SNP alleles identified alter amino acids, potentially making it easier to devise functional assays of protein function to determine pathogenicity.
Assuntos
Interação Gene-Ambiente , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Síndrome de Stevens-Johnson/genética , Síndrome de Stevens-Johnson/patologia , Alelos , Estudos de Coortes , Exoma , Genótipo , Humanos , FenótipoRESUMO
Progressive encephalopathy with oedema, hypsarrhythmia and optic atrophy (PEHO) syndrome is a rare Mendelian phenotype comprising severe retardation, early onset epileptic seizures, optic nerve/cerebellar atrophy, pedal oedema, and early death. Atypical cases are often known as PEHO-like, and there is an overlap with 'early infantile epileptic encephalopathy'. PEHO is considered to be recessive, but surprisingly since initial description in 1991, no causative recessive gene(s) have been described. Hence, we report a multiplex consanguineous family with the PEHO phenotype where affected individuals had a homozygous frame-shift deletion in CCDC88A (c.2313delT, p.Leu772*ter). Analysis of cDNA extracted from patient lymphocytes unexpectedly failed to show non-sense mediated decay, and we demonstrate that the mutation produces a truncated protein lacking the crucial C-terminal half of CCDC88A (girdin). To further investigate the possible role of CCDC88A in human neurodevelopment we re-examined the behaviour and neuroanatomy of Ccdc88a knockout pups. These mice had mesial-temporal lobe epilepsy, microcephaly and corpus callosum deficiency, and by postnatal Day 21, microcephaly; the mice died at an early age. As the mouse knockout phenotype mimics the human PEHO phenotype this suggests that loss of CCDC88A is a cause of the PEHO phenotype, and that CCDC88A is essential for multiple aspects of normal human neurodevelopment.
Assuntos
Edema Encefálico/diagnóstico , Edema Encefálico/genética , Proteínas dos Microfilamentos/genética , Mutação/genética , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Atrofia Óptica/diagnóstico , Atrofia Óptica/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética , Proteínas de Transporte Vesicular/genética , Animais , Encéfalo/patologia , Criança , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , LinhagemRESUMO
Congenital inability to feel pain is very rare but the identification of causative genes has yielded significant insights into pain pathways and also novel targets for pain treatment. We report a novel recessive disorder characterized by congenital insensitivity to pain, inability to feel touch, and cognitive delay. Affected individuals harboured a homozygous missense mutation in CLTCL1 encoding the CHC22 clathrin heavy chain, p.E330K, which we demonstrate to have a functional effect on the protein. We found that CLTCL1 is significantly upregulated in the developing human brain, displaying an expression pattern suggestive of an early neurodevelopmental role. Guided by the disease phenotype, we investigated the role of CHC22 in two human neural crest differentiation systems; human induced pluripotent stem cell-derived nociceptors and TRKB-dependant SH-SY5Y cells. In both there was a significant downregulation of CHC22 upon the onset of neural differentiation. Furthermore, knockdown of CHC22 induced neurite outgrowth in neural precursor cells, which was rescued by stable overexpression of small interfering RNA-resistant CHC22, but not by mutant CHC22. Similarly, overexpression of wild-type, but not mutant, CHC22 blocked neurite outgrowth in cells treated with retinoic acid. These results reveal an essential and non-redundant role for CHC22 in neural crest development and in the genesis of pain and touch sensing neurons.
Assuntos
Cadeias Pesadas de Clatrina/genética , Mutação/genética , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Dor/genética , Tato/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Músculo Esquelético/metabolismo , Neurônios/metabolismo , Dor/metabolismoRESUMO
Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.
Assuntos
Estrona/metabolismo , Placofilinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Citocinese/genética , Citocinese/fisiologia , Estrona/genética , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Placofilinas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.
RESUMO
Germline mutations in the FLCN gene cause Birt-Hogg-Dubé syndrome, familial spontaneous pneumothorax, or apparently nonsyndromic inherited RCC. The vast majority of reported FLCN mutations are predicted to result in a truncated/absent gene product and so infrequent missense and inframe-deletion (IFD) FLCN mutations might indicate critical functional domains. To investigate this hypothesis we (1) undertook an in silico evolutionary analysis of the FLCN sequence and (2) investigated in vitro the functional effects of naturally occurring FLCN missense/IFD mutations. The folliculin protein sequence evolved more slowly and was under stronger purifying selection than the average gene, most notably at a region between codons 100 and 230. Pathogenic missense and IFD FLCN mutations that impaired folliculin tumor suppressor function significantly disrupted the stability of the FLCN gene product but two missense substitutions initially considered to be putative mutations did not impair protein stability, growth suppression activity, or intracellular localization of folliculin. These findings are consistent with the distribution of FLCN mutations throughout the coding sequence, and suggest that multiple protein domains contribute to folliculin stability and tumor suppressor activity. In vitro assessment of protein stability and tumor suppressor activity provides a practical strategy for assessing the pathogenicity of potential FLCN mutations.
Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Proliferação de Células , Biologia Computacional , Evolução Molecular , Ordem dos Genes , Humanos , Espaço Intracelular/metabolismo , Modelos Estatísticos , Estabilidade Proteica , Transporte Proteico/genéticaRESUMO
BACKGROUND Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant multisystem disorder with skin (fibrofolliculomas or trichodiscomas), lung (cysts and pneumothorax) and kidney (renal cell carcinoma) tumours. Although colorectal neoplasia was reported initially to be part of the BHD phenotype, some recent studies have not confirmed this association. METHODS A series of clinical and laboratory studies was undertaken to investigate possible relationships between colorectal neoplasia and the BHD gene (FLCN). The studies investigated whether individuals with familial colorectal cancer of unknown cause might have unsuspected germline FLCN mutations, looked for somatic FLCN C(8) tract mutations in microsatellite unstable sporadic colorectal cancers, and assessed the risk of colorectal neoplasia and possible genotype-phenotype correlations in BHD patients. RESULTS Although it was found previously that germline FLCN mutations can be detected in approximately 5% of patients with familial renal cell carcinoma, germline FLCN mutations were not detected in 50 patients with familial non-syndromic colorectal cancer. Analysis of genotype-phenotype correlations for two recurrent FLCN mutations identified in a subset of 51 families with BHD demonstrated a significantly higher risk of colorectal neoplasia in c.1285dupC mutation (within the exon 11 C(8) mononucleotide tract) carriers than in c.610delGCinsTA mutation carriers (chi(2)=5.78, p=0.016). Somatic frameshift mutations in the FLCN exon 11 C(8) mononucleotide tract were detected in 23% of sporadic colorectal cancers with microsatellite instability, suggesting that FLCN inactivation might contribute to colorectal tumourigenesis. CONCLUSIONS These findings suggest that the previously reported clinical heterogeneity for colorectal neoplasia may reflect allelic heterogeneity and the risk of colorectal neoplasia in BHD syndrome requires further investigation.
Assuntos
Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Adulto , Idoso , Sequência de Bases , Carcinoma de Células Renais/complicações , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Cistos/complicações , Análise Mutacional de DNA , Saúde da Família , Feminino , Genótipo , Humanos , Neoplasias Renais/complicações , Pneumopatias/complicações , Masculino , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Fenótipo , Pneumotórax/complicações , Dermatopatias/complicações , SíndromeRESUMO
Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition characterised by the presence of facial fibrofolliculomas, pulmonary cysts which may be associated with spontaneous pneumothorax and renal tumours. Germline mutations in the gene Folliculin (FLCN) were first identified in BHD patients in 2002. In addition FLCN mutations have also been described in families with isolated primary spontaneous pneumothorax (PSP) and also familial clear cell renal carcinomas (FcRCC). We have established a locus-specific database based on the Leiden Open (source) Variation Database (LOVD) software. The version of the database contains 60 previously published mutations and 10 previously unpublished novel germline FLCN mutations. The mutations are comprised of deletions (44.3%), substitutions (35.7%), duplications (14.3%) and deletion/insertions (5.7%). The database is accessible online at http://www.lovd.nl/flcn.
Assuntos
Bases de Dados Genéticas , Éxons/genética , Íntrons/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Folículo Piloso/patologia , Humanos , Internet , Neoplasias Renais/genética , Pneumotórax/genética , Dermatopatias Genéticas/genética , SíndromeRESUMO
By studying healthy women who do not request analgesia during their first delivery, we investigate genetic effects on labor pain. Such women have normal sensory and psychometric test results, except for significantly higher cuff pressure pain. We find an excess of heterozygotes carrying the rare allele of SNP rs140124801 in KCNG4. The rare variant KV6.4-Met419 has a dominant-negative effect and cannot modulate the voltage dependence of KV2.1 inactivation because it fails to traffic to the plasma membrane. In vivo, Kcng4 (KV6.4) expression occurs in 40% of retrograde-labeled mouse uterine sensory neurons, all of which express KV2.1, and over 90% express the nociceptor genes Trpv1 and Scn10a. In neurons overexpressing KV6.4-Met419, the voltage dependence of inactivation for KV2.1 is more depolarized compared with neurons overexpressing KV6.4. Finally, KV6.4-Met419-overexpressing neurons have a higher action potential threshold. We conclude that KV6.4 can influence human labor pain by modulating the excitability of uterine nociceptors.
Assuntos
Dor do Parto/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Analgésicos/farmacologia , Animais , Sequência de Bases , Membrana Celular/metabolismo , Cognição , Estudos de Coortes , Emoções , Feminino , Gânglios Espinais/metabolismo , Heterozigoto , Humanos , Ativação do Canal Iônico/genética , Dor do Parto/genética , Dor do Parto/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Nociceptores/metabolismo , Limiar da Dor , Polimorfismo de Nucleotídeo Único/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Gravidez , Multimerização Proteica , Células Receptoras Sensoriais/metabolismo , Canais de Potássio Shab/metabolismo , Frações Subcelulares/metabolismo , Útero/inervaçãoRESUMO
The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active 'mature' forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.
Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Neuropeptídeos/metabolismo , Proteólise , Comunicação Autócrina , Diferenciação Celular , Linhagem Celular Tumoral , Cadeias Pesadas de Clatrina/genética , Técnicas de Silenciamento de Genes , Humanos , Lisossomos , Neurônios , Comunicação Parácrina , Transporte ProteicoRESUMO
A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection.
Assuntos
Genoma Viral , Microcefalia/metabolismo , Microcefalia/virologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Encéfalo/anormalidades , Encéfalo/metabolismo , Encéfalo/virologia , Criança , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Mutação , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/virologia , Células Vero , Zika virus/genéticaRESUMO
Erroneous activation of the pain-sensing system, as in chronic or neuropathic pain, represents a major health burden with insufficient treatment options. However, the study of genetic disorders rendering individuals completely unable to feel pain offers hope. All causes of congenital painlessness affect nociceptors, evolutionarily conserved specialist neurons able to sense all type of tissue damage. The discovery of new genes essential for sensing pain (SCN11A, PRDM12, and CLTCL1) has provided unexpected insights into the biological mechanisms that drive distinct stages of nociception. Drugs targeting two previously discovered painlessness genes, NGF and SCN9A, are currently in late-stage clinical trials; thus, characterization of these new painlessness genes has significant potential for the generation of new classes of analgesics.
Assuntos
Nociceptores/fisiologia , Percepção da Dor/fisiologia , Transtornos da Percepção/genética , Proteínas de Transporte/genética , Cadeias Pesadas de Clatrina/genética , Humanos , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Transtornos da Percepção/fisiopatologiaRESUMO
Pain perception has evolved as a warning mechanism to alert organisms to tissue damage and dangerous environments. In humans, however, undesirable, excessive or chronic pain is a common and major societal burden for which available medical treatments are currently suboptimal. New therapeutic options have recently been derived from studies of individuals with congenital insensitivity to pain (CIP). Here we identified 10 different homozygous mutations in PRDM12 (encoding PRDI-BF1 and RIZ homology domain-containing protein 12) in subjects with CIP from 11 families. Prdm proteins are a family of epigenetic regulators that control neural specification and neurogenesis. We determined that Prdm12 is expressed in nociceptors and their progenitors and participates in the development of sensory neurons in Xenopus embryos. Moreover, CIP-associated mutants abrogate the histone-modifying potential associated with wild-type Prdm12. Prdm12 emerges as a key factor in the orchestration of sensory neurogenesis and may hold promise as a target for new pain therapeutics.