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1.
J Hum Genet ; 69(1): 53-58, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37697026

RESUMO

Heterozygous deleterious variants in SKI cause Shprintzen-Goldberg Syndrome, which is mainly characterized by craniofacial features, neurodevelopmental disorder and thoracic aorta dilatations/aneurysms. The encoded protein is a member of the transforming growth factor beta signaling. Paucity of reported studies exploring the SGS molecular pathogenesis hampers disease recognition and clinical interpretation of private variants. Here, the unpublished c.349G>A, p.[Gly117Ser] and the recurrent c.539C>T, p.[Thr180Met] SKI variants were studied combining in silico and in vitro approach. 3D comparative modeling and calculation of the interaction energy predicted that both variants alter the SKI tertiary protein structure and its interactions. Computational data were functionally corroborated by the demonstration of an increase of MAPK phosphorylation levels and alteration of cell cycle in cells expressing the mutant SKI. Our findings confirmed the effects of SKI variants on MAPK and opened the path to study the role of perturbations of the cell cycle in SGS.


Assuntos
Síndrome de Marfan , Simulação de Dinâmica Molecular , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ciclo Celular/genética , Fator de Crescimento Transformador beta
2.
Haematologica ; 2024 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-38385274

RESUMO

Primary hemophagocytic lymphohistiocytosis (pHLH) is a severe, life-threatening hyperinflammatory syndrome caused by defects in genes of the granule-dependent cytotoxic pathway. Here we investigated the clinical presentation and outcome in a large cohort of 143 patients with pHLH diagnosed in the last 15 years and enrolled in the Italian registry. The median age at diagnosis was 12 months (IQR 2-81), and ninety-two patients (64%) fulfilled the HLH-2004 criteria. Out of 111 patients who received first-line combined therapy (HLH-94, HLH-2004, Euro-HIT protocols), 65 (59%) achieved complete response (CR) and 21 (19%) partial response (PR). Thereafter, 33 patients (30%) reactivated, and 92 (64%) received HSCT, 78 of whom (85%) survived and were alive at a median follow-up from diagnosis of 67 months. Thirty-six patients (25%) died before HSCT and 14 (10%) after. Overall, 93 patients (65%) were alive after a median follow-up of 30 months. Unadjusted predictors of non-response were age.

3.
Hum Genet ; 142(6): 785-808, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37079061

RESUMO

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.


Assuntos
Variação Genética , Instabilidade Articular , Humanos , Estados Unidos , Testes Genéticos/métodos , Instabilidade Articular/diagnóstico , Instabilidade Articular/genética , Análise de Sequência de DNA/métodos
4.
Pediatr Res ; 93(5): 1208-1215, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-35953512

RESUMO

BACKGROUND: Loss-of-function variants in MID1 are the most common cause of Opitz G/BBB syndrome (OS). The interpretation of intronic variants affecting the splicing is a rising issue in OS. METHODS: Exon sequencing of a 2-year-old boy with OS showed that he was a carrier of the de novo c.1286-10G>T variant in MID1. In silico predictions and minigene assays explored the effect of the variant on splicing. The minigene approach was also applied to two previously identified MID1 c.864+1G>T and c.1285+1G>T variants. RESULTS: Minigene assay demonstrated that the c.1286-10G>T variant generated the inclusion of eight nucleotides that predicted generation of a frameshift. The c.864+1G>T and c.1285+1G>T variants resulted in an in-frame deletion predicted to generate a shorter MID1 protein. In hemizygous males, this allowed reclassification of all the identified variants from "of unknown significance" to "likely pathogenic." CONCLUSIONS: Minigene assay supports functional effects from MID1 intronic variants. This paves the way to the introduction of similar second-tier investigations in the molecular diagnostics workflow of OS. IMPACT: Causative intronic variants in MID1 are rarely investigated in Opitz syndrome. MID1 is not expressed in blood and mRNA studies are hardly accessible in routine diagnostics. Minigene assay is an alternative for assessing the effect of intronic variants on splicing. This is the first study characterizing the molecular consequences of three MID1 variants for diagnostic purposes and demonstrating the efficacy of minigene assays in supporting their clinical interpretation. Review of the criteria according to the American College of Medical Genetics reassessed all variants as likely pathogenic.


Assuntos
Fissura Palatina , Hipertelorismo , Masculino , Humanos , Pré-Escolar , Mutação , Fissura Palatina/genética , Hipertelorismo/genética , Ubiquitina-Proteína Ligases/genética
5.
Hum Mol Genet ; 29(18): 3122-3131, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32959051

RESUMO

SPONASTRIME dysplasia is an ultrarare spondyloepimetaphyseal dysplasia featuring short stature and short limbs, platyspondyly, depressed nasal bridge with midface hypoplasia and striated metaphyses. In 2019, an autosomal recessive inheritance was demonstrated by the identification of bi-allelic hypomorphic alleles in TONSL. The encoded protein has a critical role in maintaining genome integrity by promoting the homologous recombination required for repairing spontaneous replication-associated DNA lesions at collapsed replication forks. We report a 9-year-old girl with typical SPONASTRIME dysplasia and resulted in carrier of the novel missense p.(Gln430Arg) and p.(Leu1090Arg) variants in TONSL at whole-exome sequencing. In silico analysis predicted that these variants induced thermodynamic changes with a pathogenic impact on protein function. To support the pathogenicity of the identified variants, cytogenetic analysis and microscopy assays showed that patient-derived fibroblasts exhibited spontaneous chromosomal breaks and flow cytometry demonstrated defects in cell proliferation and enhanced apoptosis. These findings contribute to our understanding of the molecular pathogenesis of SPONASTRIME dysplasia and might open the way to novel therapeutic approaches.


Assuntos
Quebra Cromossômica , Predisposição Genética para Doença , NF-kappa B/genética , Osteocondrodisplasias/genética , Apoptose/genética , Proliferação de Células/genética , Criança , Feminino , Citometria de Fluxo , Humanos , Sequenciamento do Exoma
6.
Genet Med ; 24(2): 439-453, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34906501

RESUMO

PURPOSE: This study aimed to describe a multisystemic disorder featuring cardiovascular, facial, musculoskeletal, and cutaneous anomalies caused by heterozygous loss-of-function variants in TAB2. METHODS: Affected individuals were analyzed by next-generation technologies and genomic array. The presumed loss-of-function effect of identified variants was assessed by luciferase assay in cells transiently expressing TAB2 deleterious alleles. In available patients' fibroblasts, variant pathogenicity was further explored by immunoblot and osteoblast differentiation assays. The transcriptomic profile of fibroblasts was investigated by RNA sequencing. RESULTS: A total of 11 individuals from 8 families were heterozygotes for a novel TAB2 variant. In total, 7 variants were predicted to be null alleles and 1 was a missense change. An additional subject was heterozygous for a 52 kb microdeletion involving TAB2 exons 1 to 3. Luciferase assay indicated a decreased transcriptional activation mediated by NF-κB signaling for all point variants. Immunoblot analysis showed a reduction of TAK1 phosphorylation while osteoblast differentiation was impaired. Transcriptomic analysis identified deregulation of multiple pleiotropic pathways, such as TGFß-, Ras-MAPK-, and Wnt-signaling networks. CONCLUSION: Our data defined a novel disorder associated with loss-of-function or, more rarely, hypomorphic alleles in a restricted linker region of TAB2. The pleiotropic manifestations in this disorder partly recapitulate the 6q25.1 (TAB2) microdeletion syndrome and deserve the definition of cardio-facial-cutaneous-articular syndrome.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , NF-kappa B , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Éxons/genética , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais
7.
Int J Mol Sci ; 24(1)2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36614110

RESUMO

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of PRKG1, SULF1, and SYDE1 genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3'-UTR of PRKG1, SULF1 and SYDE1, a bioinformatics tool was used. To test whether PRKG1, SULF1, and SYDE1 are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between PRKG1 and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3'-UTR regions of PRKG1, SULF1, and SYDE1 which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3'-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and PRKG1, SULF1, and SYDE1 expression levels was observed. Finally, a reduction of the endogenous level of PRKG1 in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the PRKG1 gene as a direct target and SULF1 and SYDE1 as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I , Acalasia Esofágica , MicroRNAs , Humanos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Acalasia Esofágica/genética , Células HEK293 , MicroRNAs/genética , MicroRNAs/metabolismo
8.
Hum Mol Genet ; 28(13): 2133-2142, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30806661

RESUMO

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.


Assuntos
Exostose Múltipla Hereditária/genética , N-Acetilglucosaminiltransferases/genética , Proliferação de Células , Sobrevivência Celular , Feminino , Estudos de Associação Genética , Complexo de Golgi/enzimologia , Células HEK293 , Humanos , Masculino , Mutação , N-Acetilglucosaminiltransferases/análise
9.
Clin Genet ; 99(6): 829-835, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33604894

RESUMO

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteína KRIT1/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Humanos , Splicing de RNA/genética , RNA Mensageiro/genética
10.
Am J Med Genet A ; 185(3): 955-965, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33369135

RESUMO

ATP6V0A2-related cutis laxa, also known as autosomal recessive cutis laxa type 2A (ARCL2A), is a subtype of hereditary cutis laxa originally characterized by skin, skeletal, and neurological involvement, and a combined defect of N-glycosylation and O-glycosylation. The associated clinical spectrum subsequently expanded to a less severe phenotype dominated by cutaneous involvement. At the moment, ARCL2A was described in a few case reports and series only. An Italian adult woman ARCL2A with a phenotype restricted to skin and the two novel c.3G>C and c.1101dup ATP6V0A2 variants has been reported. A systematic literature review allowed us to identify 69 additional individuals from 64 families. Available data were scrutinized in order to describe the clinical and molecular variability of ARCL2A. About 78.3% of known variants were predicted null alleles, while 11 were missense and 2 affected noncanonical splice sites. Age at ascertainment appeared as the unique phenotypic discriminator with earlier age more commonly associated with facial dysmorphism (p .02), high/cleft palate (p .005), intellectual disability/global developmental delay (p .013), and seizures (p .024). No specific genotype-phenotype correlations were identified. This work confirmed the existence of an attenuated phenotype associated with ATP6V0A2 biallelic variants and offers an updated critique to the clinical and molecular variability of ARCL2A.


Assuntos
Cútis Laxa/genética , ATPases Translocadoras de Prótons/genética , Adulto , Fatores Etários , Alelos , Sequência de Bases , Códon sem Sentido , Cútis Laxa/diagnóstico , Éxons/genética , Feminino , Mutação da Fase de Leitura , Genes Recessivos , Estudos de Associação Genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Expectativa de Vida , Mutação com Perda de Função , Mutação de Sentido Incorreto , Linhagem , Fenótipo , ATPases Translocadoras de Prótons/deficiência , Sítios de Splice de RNA/genética , Pele/patologia
11.
Adv Exp Med Biol ; 1348: 207-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34807421

RESUMO

Ehlers-Danlos syndrome is an umbrella term for a clinically and genetically heterogeneous group of hereditary soft connective tissue disorders mainly featuring abnormal cutaneous texture (doughy/velvety, soft, thin, and/or variably hyperextensible skin), easy bruising, and joint hypermobility. Currently, musculoskeletal manifestations related to joint hypermobility are perceived as the most prevalent determinants of the quality of life of affected individuals. The 2017 International Classification of Ehlers-Danlos syndromes and related disorders identifies 13 clinical types due to deleterious variants in 19 different genes. Recent publications point out the possibility of a wider spectrum of conditions that may be considered members of the Ehlers-Danlos syndrome community. Most Ehlers-Danlos syndromes are due to inherited abnormalities affecting the biogenesis of fibrillar collagens and other components of the extracellular matrix. The introduction of next-generation sequencing technologies in the diagnostic setting fastened patients' classification and improved our knowledge on the phenotypic variability of many Ehlers-Danlos syndromes. This is impacting significantly patients' management and family counseling. At the same time, most individuals presenting with joint hypermobility and associated musculoskeletal manifestations still remain without a firm diagnosis, due to a too vague clinical presentation and/or the lack of an identifiable molecular biomarker. These individuals are currently defined with the term "hypermobility spectrum disorders". Hence, in parallel with a continuous update of the International Classification of Ehlers-Danlos syndromes, the scientific community is investing efforts in offering a more efficient framework for classifying and, hopefully, managing individuals with joint hypermobility.


Assuntos
Síndrome de Ehlers-Danlos , Instabilidade Articular , Bases de Dados Genéticas , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/epidemiologia , Síndrome de Ehlers-Danlos/genética , Matriz Extracelular , Humanos , Instabilidade Articular/diagnóstico , Instabilidade Articular/epidemiologia , Instabilidade Articular/genética , Qualidade de Vida
12.
Int J Mol Sci ; 23(1)2021 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-35008880

RESUMO

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.


Assuntos
Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Especificidade por Substrato
14.
Int J Mol Sci ; 21(14)2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32698527

RESUMO

Transforming growth factor ß (TGF-ß) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-ß results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-ß signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-ß signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor ß (TGF-ß) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-ß signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-ß signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.


Assuntos
Contratura/patologia , Transdução de Sinais , Dermatopatias Genéticas/patologia , Pele/patologia , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Contratura/diagnóstico por imagem , Contratura/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Imageamento por Ressonância Magnética , Fosforilação , Pele/diagnóstico por imagem , Pele/metabolismo , Dermatopatias Genéticas/diagnóstico por imagem , Dermatopatias Genéticas/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1865(6): 908-919, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29604308

RESUMO

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.


Assuntos
Proteína Beclina-1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
16.
Hum Mutat ; 40(11): e24-e36, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31254430

RESUMO

Familial cerebral cavernous malformation (FCCM) is an autosomal dominant vascular disorder caused by heterozygous deleterious variants in KRIT1, CCM2 or PDCD10. In a previous study, we presented the clinical and molecular findings in 140 FCCM individuals. In the present work, we report supporting information on (a) applied diagnostic workflow; (b) clinical significance of molecular findings according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology recommendations; (c) standardization of molecular and clinical data according to the Human Phenotype Ontology; (d) preliminary genotype-phenotype correlations on a subgroup of patients by considering sex, age at diagnosis, neurological symptoms, and number and anatomical site(s) of vascular anomalies; (e) datasets submitted to the Leiden Open Variation Database. An overview of the changes of our diagnostic approach before and after the transition to next-generation sequencing is also reported. This work presents the full procedure that we apply for molecular testing, data interpretation and storing in public databases in FCCM.


Assuntos
Interpretação Estatística de Dados , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Fluxo de Trabalho , Alelos , Biologia Computacional/métodos , Bases de Dados Genéticas , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Fenótipo
17.
Hum Mutat ; 40(10): 1886-1898, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31250519

RESUMO

Transforming growth factor ß-activated kinase 1 (TAK1) mediates multiple biological processes through the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways. TAK1 activation is tightly regulated by its binding partners (TABs). In particular, binding with TAB2 is crucial for cardiovascular development and extracellular matrix (ECM) homeostasis. In our previous work, we reported a novel multisystem disorder associated with the heterozygous TAB2 c.1398dup variant. Here, we dissect the functional effects of this variant in order to understand its molecular pathogenesis. We demonstrate that TAB2 c.1398dup considerably undergoes to nonsense-mediated messenger RNA decay and encodes a truncated protein that loses its ability to bind TAK1. We also show an alteration of the TAK1 autophosphorylation status and of selected downstream signaling pathways in patients' fibroblasts. Immunofluorescence analyses and ECM-related polymerase chain reaction-array panels highlight that patient fibroblasts display ECM disorganization and altered expression of selected ECM components and collagen-related pathways. In conclusion, we deeply dissect the molecular pathogenesis of the TAB2 c.1398dup variant and show that the resulting phenotype is well explained by TAB2 loss-of-function. Our data also offer initial insights on the ECM homeostasis impairment as a molecular mechanism probably underlying a multisystem disorder linked to TAB2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Matriz Extracelular/metabolismo , Variação Genética , Haploinsuficiência , Homeostase , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Linhagem Celular , Proliferação de Células , Análise Mutacional de DNA , Fibroblastos/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Fosforilação , Ligação Proteica , Transdução de Sinais
20.
Hum Mutat ; 39(12): 1885-1900, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30161288

RESUMO

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Neoplasias do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteína KRIT1/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência , Adulto , Idoso , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas de Transporte/metabolismo , Células Cultivadas , Neoplasias do Sistema Nervoso Central/metabolismo , Criança , Pré-Escolar , Simulação por Computador , Éxons , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Itália , Proteína KRIT1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Proteínas Proto-Oncogênicas/metabolismo , Estudos Retrospectivos , Adulto Jovem
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