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BACKGROUND: Neurofibromatosis type 1 (NF1) is characterized by the highly variable and unpredictable development of benign peripheral nerve sheath tumours: cutaneous (cNFs), subcutaneous (scNFs) and plexiform (pNFs) neurofibromas. OBJECTIVES: To identify neurofibroma modifier genes, in order to develop a database of patients with NF1. METHODS: All patients were phenotypically evaluated by a medical practitioner using a standardized questionnaire and the causal NF1 variant identified. We enrolled 1333 patients with NF1 who were genotyped for > 7 million common variants. RESULTS: A genome-wide association case-only study identified a significant association with 9q21.33 in the pNF phenotype in the discovery cohort. Twelve, three and four regions suggestive of association at the P ≤ 1 × 10-6 threshold were identified for pNFs, cNFs and scNFs, respectively. Evidence of replication was observed for 4, 2 and 6 loci, including 168 candidate modifier protein-coding genes. Among the candidate modifier genes, some were implicated in the RAS-mitogen-activated protein kinase pathway, cell-cycle control and myelination. Using an original CRISPR/Cas9-based functional assay, we confirmed GAS1 and SPRED2 as pNF and scNF candidate modifiers, as their inactivation specifically affected NF1-mutant Schwann cell growth. CONCLUSIONS: Our study may shed new light on the pathogenesis of NF1-associated neurofibromas and will, hopefully, contribute to the development of personalized care for patients with this deleterious and life-threatening condition.
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Neurofibroma Plexiforme , Neurofibroma , Neurofibromatose 1 , Humanos , Neurofibromatose 1/genética , Neurofibroma Plexiforme/complicações , Neurofibroma Plexiforme/genética , Estudo de Associação Genômica Ampla , Neurofibroma/complicações , Neurofibroma/genética , Genótipo , Proteínas Repressoras/genéticaRESUMO
Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.
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Neurofibromatose 1 , Humanos , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética , Reação em Cadeia da Polimerase Multiplex , Recombinação Homóloga , Fenótipo , Família , Deleção de GenesRESUMO
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases.
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Neurofibromatose 1 , Humanos , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética , Genes da Neurofibromatose 1 , Hibridização Genômica Comparativa , Éxons , Sequenciamento Completo do GenomaRESUMO
CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. OBJECTIVE: MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654Gâ >â T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. DESIGN: Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. RESULTS: Genetic testing of the proband's unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. CONCLUSION: We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.
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Hiperparatireoidismo , Neoplasia Endócrina Múltipla Tipo 1 , Teste Pré-Natal não Invasivo , Adulto , Feminino , Testes Genéticos , Humanos , Hiperparatireoidismo/genética , Masculino , Neoplasia Endócrina Múltipla Tipo 1/genética , Herança Paterna , GravidezRESUMO
Complete deletion of the NF1 gene is identified in 5-10% of patients with neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with deletion of the NF1 locus, but comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome. NF1-deleted patients were enrolled and phenotypically characterized with a standardized questionnaire between 2005 and 2020 from a large French NF1 cohort. Statistical analyses for main NF1-associated symptoms were performed versus an NF1 reference population. A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. The median age of the group at clinical investigations was 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1-deleted patients fulfilled the NIH criteria for NF1. More than half had café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described "classic" NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1-deleted group. We described the largest NF1-deleted cohort to date and clarified the more severe phenotype observed in these patients.
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BACKGROUND: Pediatric neurofibromatosis type 1 (NF1)-associated optic pathway gliomas (OPGs) exhibit different clinico-radiological features, treatment, and outcome compared with sporadic OPGs. While NF1-associated OPGs are caused by complete loss-of-function of the NF1 gene, other genetic alterations of the RAS-MAPK pathway are frequently described in the sporadic cases. We identified a group of patients who presented OPGs with typical radiological features of NF1-associated OPGs but without the NF1 diagnostic criteria. We aim to investigate into the possible molecular mechanisms underlying this "NF1-like" pediatric OPGs presentation. METHODS: We analyzed clinico-radiological features of 16 children with NF1-like OPGs and without NF1 diagnostic criteria. We performed targeted sequencing of the NF1 gene in constitutional samples (nâ =â 16). The RAS-MAPK pathway major genes were sequenced in OPG tumor samples (nâ =â 11); BRAF FISH and IHC analyses were also performed. RESULTS: In one patient's blood and tumor samples, we identified a NF1 nonsense mutation (exon 50: c.7285C>T, p.Arg2429*) with ~8% and ~70% VAFs, respectively, suggesting a mosaic NF1 mutation limited to the brain (segmental NF1). This patient presented signs of neurodevelopmental disorder. We identified a somatic alteration of the RAS-MAPK pathway in eight tumors: four BRAF activating p.Val600Glu mutations, three BRAF:KIAA oncogenic fusions, and one putative gain-of-function complex KRAS indel inframe mutation. CONCLUSIONS: NF1-like OPGs can rarely be associated with mosaic NF1 that needs specific constitutional DNA analyses for diagnosis. Further studies are warranted to explore unknown predisposition condition leading to the NF1-like OPG presentation, particularly in patients with the association of a neurodevelopmental disorder.
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Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but high variable expressivity. NF1 is caused by loss-of-function mutations in the NF1 gene, a negative regulator of the RAS-MAPK pathway. The NF1 gene has one of the highest mutation rates in human disorders, which may explain the outbreak of independent de novo variants in the same family. Here, we report the co-occurrence of pathogenic variants in the NF1 and SPRED1 genes in six families with NF1 and Legius syndrome, using next-generation sequencing. In five of these families, we observed the co-occurrence of two independent NF1 variants. All NF1 variants were classified as pathogenic, according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. In the sixth family, one sibling inherited a complete deletion of the NF1 gene from her mother and carried a variant of unknown significance in the SPRED1 gene. This variant was also present in her brother, who was diagnosed with Legius syndrome, a differential diagnosis of NF1. This work illustrates the complexity of molecular diagnosis in a not-so-rare genetic disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Manchas Café com Leite/genética , Neurofibromatose 1/genética , Neurofibromina 1/genética , Fenótipo , Adolescente , Adulto , Manchas Café com Leite/complicações , Manchas Café com Leite/patologia , Criança , Feminino , Humanos , Masculino , Mutação , Neurofibromatose 1/complicações , Neurofibromatose 1/patologia , LinhagemRESUMO
Background: Clinical overlap between neurofibromatosis type 2 (NF2), schwannomatosis, and meningiomatosis can make clinical diagnosis difficult. Hence, molecular investigation of germline and tumor tissues may improve the diagnosis. Methods: We present the targeted next-generation sequencing (NGS) of NF2, SMARCB1, LZTR1, SMARCE1, and SUFU tumor suppressor genes, using an amplicon-based approach. We analyzed blood DNA from a cohort of 196 patients, including patients with NF2 (N = 79), schwannomatosis (N = 40), meningiomatosis (N = 12), and no clearly established diagnosis (N = 65). Matched tumor DNA was analyzed when available. Forty-seven NF2-/SMARCB1-negative schwannomatosis patients and 27 NF2-negative meningiomatosis patients were also evaluated. Results: A NF2 variant was found in 41/79 (52%) NF2 patients. SMARCB1 or LZTR1 variants were identified in 5/40 (12.5%) and 13/40 (â¼32%) patients in the schwannomatosis cohort. Potentially pathogenic variants were found in 12/65 (18.5%) patients with no clearly established diagnosis. A LZTR1 variant was identified in 16/47 (34%) NF2/SMARCB1-negative schwannomatosis patients. A SMARCE1 variant was found in 3/39 (â¼8%) meningiomatosis patients. No SUFU variant was found in the cohort. NGS was an effective and sensitive method to detect mutant alleles in blood or tumor DNA of mosaic NF2 patients. Interestingly, we identified a 4-hit mechanism resulting in the complete NF2 loss-of-function combined with SMARCB1 and LZTR1 haploinsufficiency in two-thirds of tumors from NF2 patients. Conclusions: Simultaneous investigation of NF2, SMARCB1, LZTR1, and SMARCE1 is a key element in the differential diagnosis of NF2, schwannomatosis, and meningiomatosis. The targeted NGS strategy is suitable for the identification of NF2 mosaicism in blood and for the investigation of tumors from these patients.
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Genes Supressores de Tumor , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Meníngeas/diagnóstico , Meningioma/diagnóstico , Mutação , Neurilemoma/diagnóstico , Neurofibromatoses/diagnóstico , Neurofibromatose 2/diagnóstico , Neoplasias Cutâneas/diagnóstico , Biomarcadores Tumorais , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Diagnóstico Diferencial , Seguimentos , Humanos , Neoplasias Meníngeas/genética , Meningioma/genética , Neurilemoma/genética , Neurofibromatoses/genética , Neurofibromatose 2/genética , Neurofibromina 2/genética , Prognóstico , Estudos Prospectivos , Proteínas Repressoras/genética , Estudos Retrospectivos , Proteína SMARCB1/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição/genéticaRESUMO
Variants in KMT2A, encoding the histone methyltransferase KMT2A, are a growing cause of intellectual disability (ID). Up to now, the majority of KMT2A variants are non-sense and frameshift variants causing a typical form of Wiedemann-Steiner syndrome. We studied KMT2A gene in a cohort of 200 patients with unexplained syndromic and non-syndromic ID and identified four novel variants, one splice and three missense variants, possibly deleterious. We used primary cells from the patients and molecular approaches to determine the deleterious effects of those variants on KMT2A expression and function. For the putative splice variant c.11322-1G>A, we showed that it led to only one nucleotide deletion and loss of the C-terminal part of the protein. For two studied KMT2A missense variants, c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)), located at the cysteine-rich CXXC domain and the transactivation domain of the protein, respectively, we found altered KMT2A target genes expression in patient's fibroblasts compared to controls. Furthermore, we found a disturbed subcellular distribution of KMT2A for the c.3460C>T mutant. Taken together, our results demonstrated the deleterious impact of the splice variant and of the missense variants located at two different functional domains and suggested reduction of KMT2A function as the disease-causing mechanism.
Assuntos
Anormalidades Múltiplas/genética , Histona-Lisina N-Metiltransferase/genética , Hipertricose/genética , Deficiência Intelectual/genética , Proteína de Leucina Linfoide-Mieloide/genética , Anormalidades Múltiplas/patologia , Animais , Células COS , Células Cultivadas , Pré-Escolar , Chlorocebus aethiops , Feminino , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Hipertricose/patologia , Lactente , Deficiência Intelectual/patologia , Mutação com Perda de Função , Masculino , Mutação de Sentido Incorreto , Proteína de Leucina Linfoide-Mieloide/metabolismo , Splicing de RNA , SíndromeRESUMO
The commonest tumors associated with neurofibromatosis type 1 (NF1) are benign peripheral nerve sheath tumors, called neurofibromas. Malignant transformation of neurofibromas into aggressive MPNSTs may occur with a poor patient prognosis. A cooperative role of SUZ12 or EED inactivation, along with NF1, TP53, and CDKN2A loss-of-function, has been proposed to drive progression to MPNSTs. An exome sequencing analysis of eight MPNSTs, one plexiform neurofibroma, and seven cutaneous neurofibromas was undertaken. Biallelic inactivation of the NF1 gene was observed in the plexiform neurofibroma and the MPNSTs, underlining that somatic biallelic NF1 inactivation is likely to be the initiating event for plexiform neurofibroma genesis, although it is unlikely to be sufficient for the subsequent MPNST development. The majority (5/8) of MPNSTs in our analyses demonstrated homozygous or heterozygous deletions of CDKN2A, which may represent an early event following NF1 LOH in the malignant transformation of Schwann cells from plexiform neurofibroma to MPNST. Biallelic somatic alterations of SUZ12 was also found in 4/8 MPNSTs. EED biallelic alterations were detected in 2 of the other four MPNSTs, with one tumor having a homozygous EED deletion. A missense mutation in the chromatin regulator KDM2B was also identified in one MPNST. No TP53 point mutations were found in this study, confirming previous data that TP53 mutations may be relatively rare in NF1-associated MPNSTs. Our study confirms the frequent biallelic inactivation of PRC2 subunits SUZ12 and EED in MPNSTs, and suggests the implication of KDM2B.
Assuntos
Biomarcadores Tumorais/genética , Mutação/genética , Neoplasias de Bainha Neural/genética , Neurofibromatose 1/genética , Neurofibromina 1/genética , Proteínas F-Box/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Neoplasias , Estadiamento de Neoplasias , Complexo Repressor Polycomb 2/genética , Prognóstico , Fatores de TranscriçãoRESUMO
RAS/MAPK pathway germline mutations were described in Rasopathies, a class of rare genetic syndromes combining facial abnormalities, heart defects, short stature, skin and genital abnormalities, and mental retardation. The majority of the mutations identified in the Rasopathies are point mutations which increase RAS/MAPK pathway signaling. Duplications encompassing RAS/MAPK pathway genes (PTPN11, RAF1, MEK2, or SHOC2) were more rarely described. Here we report, a syndromic familial case of a 12p duplication encompassing the dosage sensitive gene KRAS, whose phenotype overlapped with rasopathies. The patient was referred because of a history of mild learning disabilities, small size, facial dysmorphy, and pigmentation abnormalities (café-au-lait and achromic spots, and axillar lentigines). This phenotype was reminiscent of rasopathies. No mutation was identified in the most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1. The patient constitutional DNA exhibited a ~10.5 Mb duplication at 12p, including the KRAS gene. The index case's mother carried the same chromosome abnormality and also showed development delay with short stature, and numerous café-au-lait spots. Duplication of the KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies.
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Variações do Número de Cópias de DNA/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adolescente , Manchas Café com Leite/genética , Criança , Cromossomos Humanos Par 12/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase 2/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas c-raf/genéticaRESUMO
BACKGROUND: Heterozygous NSD1 mutations were identified in 60%-90% of patients with Sotos syndrome. Recently, mutations of the SETD2 and DNMT3A genes were identified in patients exhibiting only some Sotos syndrome features. Both NSD1 and SETD2 genes encode epigenetic 'writer' proteins that catalyse methylation of histone 3 lysine 36 (H3K36me). The DNMT3A gene encodes an epigenetic 'reader' protein of the H3K36me chromatin mark. METHODS: We aimed at confirming the implication of DNMT3A and SETD2 mutations in an overgrowth phenotype, through a comprehensive targeted-next generation sequencing (NGS) screening in 210 well-phenotyped index cases with a Sotos-like phenotype and no NSD1 mutation, from a French cohort. RESULTS: Six unreported heterozygous likely pathogenic variants in DNMT3A were identified in seven patients: two nonsense variants and four de novo missense variants. One de novo unreported heterozygous frameshift variant was identified in SETD2 in one patient. All the four DNMT3A missense variants affected DNMT3A functional domains, suggesting a potential deleterious impact. DNMT3A-mutated index cases shared similar clinical features including overgrowth phenotype characterised by postnatal tall stature (≥+2SD), macrocephaly (≥+2SD), overweight or obesity at older age, intellectual deficiency and minor facial features. The phenotype associated with SETD2 mutations remains to be described more precisely. The p.Arg882Cys missense de novo constitutional DNMT3A variant found in two patients is the most frequent DNMT3A somatic mutation in acute leukaemia. CONCLUSIONS: Our results illustrate the power of targeted NGS to identify rare disease-causing variants. These observations provided evidence for a unifying mechanism (disruption of apposition and reading of the epigenetic chromatin mark H3K36me) that causes an overgrowth syndrome phenotype. Further studies are needed in order to assess the role of SETD2 and DNMT3A in intellectual deficiency without overgrowth.
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Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of â¼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with â¼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.
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Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética , Proteínas Adaptadoras de Transdução de Sinal , Biologia Computacional/métodos , Éxons , Duplicação Gênica , Genes da Neurofibromatose 1 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Mosaicismo , Mutação , Reprodutibilidade dos Testes , Estudos Retrospectivos , Deleção de SequênciaRESUMO
Sub1 and Maf1 exert an opposite effect on RNA polymerase III transcription interfering with different steps of the transcription cycle. In this study, we present evidence that Sub1 and Maf1 also exhibit an opposite role on yeast chronological life span. First, cells lacking Sub1 need more time than wild type to exit from resting and this lag in re-proliferation is correlated with a delay in transcriptional reactivation. Second, our data show that the capacity of the cells to properly establish a quiescent state is impaired in the absence of Sub1 resulting in a premature death that is dependent on the Ras/PKA and Tor1/Sch9 signalling pathways. On the other hand, we show that maf1Δ cells are long-lived mutant suggesting a connection between Pol III transcription and yeast longevity.
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Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proliferação de Células , Sobrevivência Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Longevidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Ativação Transcricional , Proteínas ras/metabolismoRESUMO
Goldberg-Shprintzen syndrome (GOSHS, MIM #609460) is an autosomal recessive disorder of intellectual disability, specific facial gestalt and Hirschsprung's disease (HSCR). In 2005, homozygosity mapping in a large consanguineous family identified KIAA1279 as the disease-causing gene. KIAA1279 encodes KIF-binding protein (KBP), whose function is incompletely understood. Studies have identified either the mitochondria or the cytoskeleton as the site of KBP localization and interactions. To better delineate the KIAA1279-related clinical spectrum and the molecular mechanisms involved in GOSHS, we studied five new patients from three different families. The homozygous KIAA1279 mutations in these patients (p.Arg90X, p.Ser200X or p.Arg202IlefsX2) led to nonsense-mediated mRNA decay and loss of KBP function. Despite the absence of functional KBP, respiratory chain complex activity in patient fibroblasts was normal. KBP did not co-localize with mitochondria in control human fibroblasts, but interacted with the actin and tubulin cytoskeleton. KBP expression directly affected neurite growth in a neuron-like cell line (human neuroblastoma SH-SY5Y), in keeping with the central (polymicrogyria) and enteric (HSCR) neuronal developmental defects seen in GOSHS patients. The KBP interactions with actin filaments and microtubules (MTs) demonstrated in our study constitute the first evidence that an actin MT cross-link protein is involved in neuronal development in humans.
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Anormalidades Craniofaciais/metabolismo , Doença de Hirschsprung/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Actinas/genética , Actinas/metabolismo , Adolescente , Adulto , Criança , Anormalidades Craniofaciais/genética , Feminino , França , Doença de Hirschsprung/genética , Humanos , Lactente , Iraque , Masculino , Microtúbulos/genética , Mutação , Proteínas do Tecido Nervoso/genética , Linhagem , Ligação Proteica , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , População Branca/genéticaRESUMO
BACKGROUND: Kallmann syndrome (KS) is a genetic disorder associating pubertal failure with congenitally absent or impaired sense of smell. KS is related to defective neuronal development affecting both the migration of olfactory nerve endings and GnRH neurons. The discovery of several genetic mutations responsible for KS led to the identification of signaling pathways involved in these processes, but the mutations so far identified account for only 30% of cases of KS. Here, we attempted to identify new genes responsible for KS by using a pan-genomic approach. METHODS: From a cohort of 120 KS patients, we selected 48 propositi with no mutations in known KS genes. They were analyzed by comparative genomic hybridization array, using Agilent 105K oligonucleotide chips with a mean resolution of 50 kb. RESULTS: One propositus was found to have a heterozygous deletion of 213 kb at locus 7q21.11, confirmed by real-time qPCR, deleting 11 of the 17 SEMA3A exons. This deletion cosegregated in the propositus' family with the KS phenotype, that was transmitted in autosomal dominant fashion and was not associated with other neurological or non-neurological clinical disorders. SEMA3A codes for semaphorin 3A, a protein that interacts with neuropilins. Mice lacking semaphorin 3A expression have been showed to have a Kallmann-like phenotype. CONCLUSIONS: SEMA3A is therefore a new gene whose loss-of-function is involved in KS. These findings validate the specific role of semaphorin 3A in the development of the olfactory system and in neuronal control of puberty in humans.