RESUMO
BACKGROUND: Genetic etiologies of autism spectrum disorders (ASD) are complex, and the genetic factors identified so far are very diverse. In complex genetic diseases such as ASD, de novo or inherited chromosomal abnormalities are valuable findings for researchers with respect to identifying the underlying genetic risk factors. With gene mapping studies on these chromosomal abnormalities, dozens of genes have been associated with ASD and other neurodevelopmental genetic diseases. In the present study, we aimed to idenitfy the causative genetic factors in patients with ASD who have an apparently balanced chromosomal translocation in their karyotypes. METHODS: For mapping the broken genes as a result of chromosomal translocations, we performed whole genome DNA sequencing. Chromosomal breakpoints and large DNA copy number variations (CNV) were determined after genome alignment. Identified CNVs and single nucleotide variations (SNV) were evaluated with VCF-BED intersect and Gemini tools, respectively. A targeted resequencing approach was performed on the JMJD1C gene in all of the ASD cohorts (220 patients). For molecular modeling, we used a homology modeling approach via the SWISS-MODEL. RESULTS: We found that there was no contribution of the broken genes or regulator DNA sequences to ASD, whereas the SNVs on the JMJD1C, CNKSR2 and DDX11 genes were the most convincing genetic risk factors for underlying ASD phenotypes. CONCLUSIONS: Genetic etiologies of ASD should be analyzed comprehensively by taking into account of the all chromosomal structural abnormalities and de novo or inherited CNV/SNVs with all possible inheritance patterns.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transtorno do Espectro Autista/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Oxirredutases N-Desmetilantes/genética , Translocação Genética/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Transtorno do Espectro Autista/patologia , Aberrações Cromossômicas , Quebra Cromossômica , RNA Helicases DEAD-box/química , Variações do Número de Cópias de DNA/genética , DNA Helicases/química , Feminino , Predisposição Genética para Doença , Genoma Humano/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Masculino , Oxirredutases N-Desmetilantes/química , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Conformação Proteica , Alinhamento de SequênciaRESUMO
Bartsocas-Papas syndrome (BPS) is a rare autosomal recessive disorder characterized by popliteal pterygia, syndactyly, ankyloblepharon, filiform bands between the jaws, cleft lip and palate, and genital malformations. Most of the BPS cases reported to date are fatal either in the prenatal or neonatal period. Causative genetic defects of BPS were mapped on the RIPK4 gene encoding receptor-interacting serine/threonine kinase 4, which is critical for epidermal differentiation and development. RIPK4 variants are associated with a wide range of clinical features ranging from milder ectodermal dysplasia to severe BPS. Here, we evaluated a consanguineous Turkish family, who had two pregnancies with severe multiple malformations compatible with BPS phenotype. In order to identify the underlying genetic defect, direct sequencing of the coding region and exon-intron boundaries of RIPK4 was carried out. A homozygous transversion (c.481G>C) that leads to the substitution of a conserved aspartic acid to histidine (p.Asp161His) in the kinase domain of the protein was detected. Pathogenicity predictions, molecular modeling, and cell-based functional assays showed that Asp161 residue is required for the kinase activity of the protein, which indicates that the identified variant is responsible for the severe BPS phenotype in the family.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Anormalidades do Olho/genética , Dedos/anormalidades , Articulação do Joelho/anormalidades , Joelho/anormalidades , Deformidades Congênitas das Extremidades Inferiores/genética , Proteínas Serina-Treonina Quinases/genética , Anormalidades da Pele/genética , Sindactilia/genética , Anormalidades Urogenitais/genética , Anormalidades Múltiplas/epidemiologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Feto Abortado/patologia , Fenda Labial/epidemiologia , Fenda Labial/patologia , Fissura Palatina/epidemiologia , Fissura Palatina/patologia , Exoma/genética , Anormalidades do Olho/epidemiologia , Anormalidades do Olho/patologia , Feminino , Dedos/patologia , Predisposição Genética para Doença , Homozigoto , Humanos , Recém-Nascido , Joelho/patologia , Articulação do Joelho/patologia , Deformidades Congênitas das Extremidades Inferiores/epidemiologia , Deformidades Congênitas das Extremidades Inferiores/patologia , Mutação/genética , Fosforilação , Gravidez , Anormalidades da Pele/epidemiologia , Anormalidades da Pele/patologia , Sindactilia/epidemiologia , Sindactilia/patologia , Anormalidades Urogenitais/epidemiologia , Anormalidades Urogenitais/patologiaRESUMO
Receptor-interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor-ß 1 (TGF-ß1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF-ß signaling during epidermal homeostasis-related events and suppression of RIPK4 expression by TGF-ß1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor-κB (NF-κB), protein kinase C (PKC), wingless-type MMTV integration site family (Wnt), and (mitogen-activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad-mediated TGF-ß1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad-mediated TGF-ß1 signaling events through suppression of TGF-ß1-induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3-Smad4 interaction, nuclear localization, and TGF-ß1-induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound-scratch assay demonstrated that RIPK4 suppressed TGF-ß1-mediated wound healing through blocking TGF-ß1-induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF-ß1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF-ß1 signaling.
Assuntos
Queratinócitos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular , Movimento Celular , Humanos , Transdução de Sinais , CicatrizaçãoRESUMO
Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.
Assuntos
Bacteriófagos , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Humanos , Staphylococcus aureus , AntibacterianosRESUMO
In this study, we investigated the in vitro potential of axially 1-morpholiniumpropan-2-ol disubstituted silicon (IV) phthalocyanine (SiPc) which was synthesized previously, on HCT-116 cells as a photodynamic therapy (PDT) agent. The singlet oxygen and photodegradation quantum yields of SiPc were calculated using UV-vis spectrophotometer. The cytotoxic and phototoxic effects of SiPc were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Annexin V-FITC/PI double staining kit, cell cycle kit, and mitochondria membrane potential (ΔΨm) assay kit with JC-1 were used to indicate the cell death pathway. Caspase-3 and ß-catenin protein expressions were evaluated by western blotting. The singlet oxygen and photodegradation quantum yields of SiPc were calculated as 0.73 and 3.64 × 10-4 in DMSO. The cell viability assays showed that IC50 value of SiPc did not reach to 100 µM without irradiation. However, excellent phototoxicity was observed in the presence of SiPc upon light irradiation. The cells undergoing early/late apoptosis significantly increased in the presence SiPc at 5 µM upon light irradiation. Besides, the proportion of cells at S and G2/M phase increased. Moreover, mitochondria membrane potentials significantly decreased at 1 and 5 µM of SiPc with light irradiation. While caspase-3 expression increased, ß-catenin expression significantly decreased on HCT-116 in the presence of SiPc (p < 0.01). The results indicated that the PDT could be related to apoptosis and Wnt/ß-catenin signaling pathway. Based on our findings, SiPc exhibited a significant PDT effect on HCT-116 cells therefore, worthy of more detailed study.
Assuntos
Fotoquimioterapia , Apoptose , Células HCT116 , Humanos , Indóis/farmacologia , Isoindóis , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêuticoRESUMO
The epidermis, the outer layer of the skin, is formed by stratified keratinocyte layers. The self-renewal of the epidermis is provided by sustained proliferation and differentiation of the keratinocyte stem cells localized to the basal layer of the epidermis. Receptor-interacting protein kinase 4 (RIPK4) is an important regulator of keratinocyte differentiation, mutations of which are associated with congenital ectodermal malformations. In an attempt to identify the molecular basis of RIPK4's function, we applied yeast two-hybrid screen (Y2H) and found basal layer-specific keratin filament component keratin 14 (KRT14) as a novel RIPK4-interacting partner. During keratinocyte differentiation, layer-specific keratin composition is tightly regulated. Likewise, the basal layer specific KRT14/keratin 5 (KRT5) heterodimers are replaced by keratin 1 (KRT1)/keratin 10 (KRT10) in suprabasal layers. The regulation of keratin turnover is under the control of signaling associated with posttranslational modifications in which phosphorylation plays a major role. In this study, we verified the KRT14-RIPK4 interaction, which was identified with Y2H, in mammalian cells and showed that the interaction was direct by using proteins expressed in bacteria. According to our results, the N-terminal kinase domain of RIPK4 is responsible for KRT14-RIPK4 interaction; however, the RIPK4 kinase activity is dispensable for the interaction. In accordance with their interaction, RIPK4 and KRT14 colocalize within the cells, particularly at keratin filaments associated with perinuclear ring-like structures. Moreover, RIPK4 did not show any effect on KRT14/KRT5 heterodimer formation. Our results suggest that RIPK4 may regulate the keratin turnover required for keratinocyte differentiation through interacting with KRT14.
RESUMO
Microcephalic primordial dwarfism (MPD) is a group of autosomal recessive inherited single-gene disorders with intrauterine and postnatal global growth failure. Seckel syndrome is the most common form of the MPD. Ten genes are known with Seckel syndrome. Using genome-wide SNP genotyping and homozygosity mapping we mapped a Seckel syndrome gene to chromosomal region 4q28.1-q28.3 in a Turkish family. Direct sequencing of PLK4 (polo-like kinase 4) revealed a homozygous splicing acceptor site transition (c.31-3 A>G) that results in a premature translation termination (p.[=,Asp11Profs*14]) causing deletion of all known functional domains of the protein. PLK4 is a master regulator of centriole biogenesis and its deficiency has recently been associated with Seckel syndrome. However, the role of PLK4 in genomic stability and the DNA damage response is unclear. Evaluation of the PLK4-Seckel fibroblasts obtained from patient revealed the expected impaired centriole biogenesis, disrupted mitotic morphology, G2/M delay, and extended cell doubling time. Analysis of the PLK4-Seckel cells indicated that PLK4 is also essential for genomic stability and DNA damage response. These findings provide mechanistic insight into the pathogenesis of the severe growth failure associated with PLK4-deficiency.
Assuntos
Centrossomo/metabolismo , Dano ao DNA , Nanismo/genética , Microcefalia/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Adulto , Células Cultivadas , Criança , Pré-Escolar , Cromossomos Humanos Par 4/genética , Nanismo/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Instabilidade Genômica , Humanos , Lactente , Masculino , Microcefalia/patologia , Mitose , Linhagem , Splicing de RNA/genéticaRESUMO
Leukoencephalopathy with thalamus and brainstem involvement, and high lactate (LTBL) is a recently identified disease related to mutations in the EARS2 gene encoding glutamyl-tRNA synthetase. We report clinical and radiological findings for two siblings with new pathogenic mutations in the EARS2 gene. Both patients showed symptoms of mild-type disease, but there were clinical differences between the two siblings. While the older brother had hypotonia and delayed developmental milestones, the younger brother had seizures and spasticity in the lower extremities. Brain magnetic resonance imaging (MRI) findings were quite similar for the two siblings. MRI findings were specific to LTBL. MRI lesions of the older sibling had regressed over time. Clinical and radiological improvement, as in the previously reported patients with LTBL, may be an important clue for diagnosis.
Assuntos
Tronco Encefálico/diagnóstico por imagem , Glutamato-tRNA Ligase/genética , Ácido Láctico/sangue , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Tálamo/diagnóstico por imagem , Biomarcadores/sangue , Criança , Humanos , Lactente , Leucoencefalopatias/sangue , Imageamento por Ressonância Magnética , Masculino , Mutação , IrmãosRESUMO
AIM: This study aimed to identify the underlying genetic defect responsible for anophthalmia/microphthalmia. METHODS: In total, two Turkish families with a total of nine affected individuals were included in the study. Affymetrix 250â K single nucleotide polymorphism genotyping and homozygosity mapping were used to identify the localisation of the genetic defect in question. Coding region of the ALDH1A3 gene was screened via direct sequencing. cDNA samples were generated from primary fibroblast cell cultures for expression analysis. Reverse transcriptase PCR (RT-PCR) analysis was performed using direct sequencing of the obtained fragments. RESULTS: The causative genetic defect was mapped to chromosome 15q26.3. A homozygous G>A substitution (c.666G>A) at the last nucleotide of exon 6 in the ALDH1A3 gene was identified in the first family. Further cDNA sequencing of ALDH1A3 showed that the c.666G>A mutation caused skipping of exon 6, which predicted in-frame loss of 43 amino acids (p.Trp180_Glu222del). A novel missense c.1398C>A mutation in exon 12 of ALDH1A3 that causes the substitution of a conserved asparagine by lysine at amino acid position 466 (p.Asn466Lys) was observed in the second family. No extraocular findings-except for nevus flammeus in one affected individual and a variant of Dandy-Walker malformation in another affected individual-were observed. Autistic-like behaviour and mental retardation were observed in three cases. CONCLUSIONS: In conclusion, novel ALDH1A3 mutations identified in the present study confirm the pivotal role of ALDH1A3 in human eye development. Autistic features, previously reported as an associated finding, were considered to be the result of social deprivation and inadequate parenting during early infancy in the presented families.
Assuntos
Aldeído Oxirredutases/genética , Anoftalmia/genética , Microftalmia/genética , Mutação de Sentido Incorreto , Sítios de Splice de RNA , Adolescente , Sequência de Bases , Criança , Cromossomos Humanos Par 15/genética , Análise Mutacional de DNA , Feminino , Genes Recessivos/genética , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessively inherited disorders characterized by impaired production of adrenal steroids. Approximately 95% of all CAH are caused by mutations of the CYP21A2 that encodes 21-hydroxylase. In this study, mutation analyses of CYP21A2 were performed in 48 CAH patients from 45 Turkish families with the clinical diagnosis of 21-hydroxylase deficiency (21OHD). While in 39 (86.7%) of 21OHD patients, disease causing CYP21A2 mutations were identified in both alleles, in two 21OHD patients CYP21A2 mutations were identified only in one allele. In four patients, mutation was not detected at all. In total, seventeen known and one novel, disease causing CYP21A2 mutations were observed. Among identified mutations, previously described c.293-13C/A>G, large rearrangements and p.Q319X mutations were the most common mutations accounting for 33.3%, 14.4% and 12.2% of all evaluated chromosomes, respectively. In six families (13.3%) a novel founder mutation, c.2T>C (p.M1?), inactivating the translation initiation codon was found. This mutation is not present in pseudogene CYP21A1P and causes the classical form of the disease in six patients. In addition, depending on the nature of the rearrangements CYP21A1P/CYP21A2 chimeras were further classified as CH(c/d), and CH-1(c) was shown to be the most prominent chimera in our study group. In conclusion, with this study we identified a novel founder CYP21A2 mutation and suggest a further classification for CYP21A1P/CYP21A2 chimeras depending on the combination of junction site position and whether it is occurred as a result of deletion or conversion. Absence of disease causing mutation of CYP21A2 in ten of screened ninety chromosomes suggests the contribution of regulatory elements in occurrences of CAH due to the 21OHD.