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1.
Am J Med Genet A ; 179(10): 2049-2055, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400068

RESUMO

Histone Gene Cluster 1 Member E, HIST1H1E, encodes Histone H1.4, is one of a family of epigenetic regulator genes, acts as a linker histone protein, and is responsible for higher order chromatin structure. HIST1H1E syndrome (also known as Rahman syndrome, OMIM #617537) is a recently described intellectual disability (ID) syndrome. Since the initial description of five unrelated individuals with three different heterozygous protein-truncating variants (PTVs) in the HIST1H1E gene in 2017, we have recruited 30 patients, all with HIST1H1E PTVs that result in the same shift in frame and that cluster to a 94-base pair region in the HIST1H1E carboxy terminal domain. The identification of 30 patients with HIST1H1E variants has allowed the clarification of the HIST1H1E syndrome phenotype. Major findings include an ID and a recognizable facial appearance. ID was reported in all patients and is most frequently of moderate severity. The facial gestalt consists of a high frontal hairline and full lower cheeks in early childhood and, in later childhood and adulthood, affected individuals have a strikingly high frontal hairline, frontal bossing, and deep-set eyes. Other associated clinical features include hypothyroidism, abnormal dentition, behavioral issues, cryptorchidism, skeletal anomalies, and cardiac anomalies. Brain magnetic resonance imaging (MRI) is frequently abnormal with a slender corpus callosum a frequent finding.

2.
Am J Hum Genet ; 104(5): 957-967, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31006512

RESUMO

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.

4.
Am J Hum Genet ; 102(3): 468-479, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29429572

RESUMO

Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity.

6.
Acta Biomater ; 66: 166-176, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29128540

RESUMO

Hydrogels have shown great potential for cartilage tissue engineering applications due to their capability to encapsulate cells within biomimetic, 3-dimensional (3D) microenvironments. However, the multi-step fabrication process that is necessary to produce cell/scaffold constructs with defined dimensions, limits their off-the-shelf translational usage. In this study, we have developed a hybrid scaffolding system which combines a thermosensitive hydrogel, poly(ethylene glycol)-poly(N-isopropylacrylamide) (PEG-PNIPAAm), with a biodegradable polymer, poly(ε-caprolactone) (PCL), into a composite, electrospun microfibrous structure. A judicious optimization of material composition and electrospinning process produced a structurally self-supporting hybrid scaffold. The reverse thermosensitivity of PEG-PNIPAAm allowed its dissolution/hydration upon cell seeding within a network of PCL microfibers while maintaining the overall scaffold shape at room temperature. A subsequent temperature elevation to 37 °C induced the hydrogel's phase transition to a gel state, effectively encapsulating cells in a 3D hydrogel without the use of a mold. We demonstrated that the hybrid scaffold enhanced chondrogenic differentiation of human mesenchymal stem cells (hMSCs) based on chondrocytic gene and protein expression, which resulted in superior viscoelastic properties of the cell/scaffold constructs. The hybrid scaffold enables a facile, single-step cell seeding process to inoculate cells within a 3D hydrogel with the potential for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: Hydrogels have demonstrated the excellent ability to enhance chondrogenesis of stem cells due to their hydrated fibrous nanostructure providing a cellular environment similar to native cartilage. However, the necessity for multi-step processes, including mixing of hydrogel precursor with cells and subsequent gelation in a mold to form a defined shape, limits their off-the-shelf usage. In this study, we developed a hybrid scaffold by combining a thermosensitive hydrogel with a mechanically stable polymer, which provides a facile means to inoculate cells in a 3D hydrogel with a mold-less, single step cell seeding process. We further showed that the hybrid scaffold enhanced chondrogenesis of mesenchymal stem cells, demonstrating its potential for cartilage tissue engineering.


Assuntos
Condrogênese , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Mesenquimais/citologia , Temperatura Ambiente , Engenharia Tecidual/métodos , Tecidos Suporte/química , Resinas Acrílicas/química , Diferenciação Celular , Forma Celular , Sobrevivência Celular , Humanos , Poliésteres/química , Polietilenoglicóis/química
7.
Eur J Hum Genet ; 26(1): 64-74, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29180823

RESUMO

Whole-gene duplications and missense variants in the HUWE1 gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1 variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G > A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1 missense variants may cause syndromic ID in both males and females.

9.
Stem Cell Reports ; 8(5): 1329-1339, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28457888

RESUMO

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation.


Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Pluripotentes Induzidas/citologia , Imagem Óptica/métodos , Técnicas de Microbalança de Cristal de Quartzo/métodos , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Biológicos , Imagem Óptica/instrumentação , Técnicas de Microbalança de Cristal de Quartzo/instrumentação
10.
Eur J Hum Genet ; 25(5): 552-559, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28327570

RESUMO

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function.


Assuntos
Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Anormalidades Múltiplas/diagnóstico , Células Cultivadas , Criança , Códon de Terminação/genética , Exoma , Feminino , Heterozigoto , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Microcefalia/diagnóstico , Fenótipo , Síndrome
12.
Am J Med Genet A ; 170(11): 2835-2846, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27667800

RESUMO

KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by mutations in ANKRD11, one of the ankyrin repeat-containing cofactors. We describe 32 KBG patients aged 2-47 years from 27 families ascertained via two pathways: targeted ANKRD11 sequencing (TS) in a group who had a clinical diagnosis of KBG and whole exome sequencing (ES) in a second group in whom the diagnosis was unknown. Speech delay and learning difficulties were almost universal and variable behavioral problems frequent. Macrodontia of permanent upper central incisors was seen in 85%. Other clinical features included short stature, conductive hearing loss, recurrent middle ear infection, palatal abnormalities, and feeding difficulties. We recognized a new feature of a wide anterior fontanelle with delayed closure in 22%. The subtle facial features of KBG syndrome were recognizable in half the patients. We identified 20 ANKRD11 mutations (18 novel: all truncating) confirmed by Sanger sequencing in 32 patients. Comparison of the two ascertainment groups demonstrated that facial/other typical features were more subtle in the ES group. There were no conclusive phenotype-genotype correlations. Our findings suggest that mutation of ANKRD11 is a common Mendelian cause of developmental delay. Affected patients may not show the characteristic KBG phenotype and the diagnosis is therefore easily missed. We propose updated diagnostic criteria/clinical recommendations for KBG syndrome and suggest that inclusion of ANKRD11 will increase the utility of gene panels designed to investigate developmental delay. © 2016 The Authors. American Journal of Medical Genetics Part A Published by Wiley Periodicals, Inc.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/diagnóstico , Doenças do Desenvolvimento Ósseo/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Anormalidades Dentárias/diagnóstico , Anormalidades Dentárias/genética , Deleção Cromossômica , Cromossomos Humanos Par 16 , Hibridização Genômica Comparativa , Facies , Feminino , Humanos , Masculino , Fenótipo , Proteínas Repressoras/genética
13.
Adv Healthc Mater ; 5(12): 1524, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27333402

RESUMO

Electrospun scaffolds provide soft nanofibrous networks pliable by human induced pluripotent stem cells. J. Nam and co-workers show on page 1408 that such compliant scaffolding leads to the formation of stem cell colonies with a distinctive three-dimensional morphology. The morphological modulation resulted in the lineage-specific differentiation, suggesting a potential means to enhance translational applications of the stem cells.

14.
Adv Healthc Mater ; 5(12): 1408-12, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27187808

RESUMO

Electrospun scaffolds with varied stiffness promote distinct colony morphology of human induced pluripotent stem cells, which affects their subsequent differentiation. On soft scaffolds, induced pluripotent stem cells develop 3D colonies due to the pliability of the electrospun fibrous networks, leading to greater differentiation tendency to ectodermal lineage.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Tecidos Suporte/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
15.
Biomaterials ; 50: 10-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25736491

RESUMO

The development of xeno-free, chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard, various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal, i.e., proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly, when surface chemistry of the substrates was uniformly controlled by collagen conjugation, the stiffness of substrate was inversely related to the sphericity, a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture, implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall, we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal.


Assuntos
Técnicas de Cultura de Células/métodos , Autorrenovação Celular , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Pluripotentes Induzidas/citologia , Fenômenos Biomecânicos , Células Cultivadas , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Masculino , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo
17.
Nanotechnology ; 25(11): 115501, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24561641

RESUMO

Electrospinning was utilized to synthesize a polyaniline (PANI)/poly(ε-caprolactone) (PCL) composite in the form of nanofibers to examine its gas sensing performance. Electrical conductivity of the composite nanofibers was tailored by secondary doping with protonic acids including hydrochloride (HCl) or camphorsulfonic acid (HCSA). FT-IR and diffuse reflectance UV-vis spectroscopy were utilized to examine doping-dependent changes in the chemical structure and the protonation state of the nanofibers, respectively. The oxidation and protonation state of the composite nanofibers were shown to strongly depend on the doping agent and duration, demonstrating a simple way of controlling the electrical conductivity of the composite. PANI/PCL electrospun nanofibers having various electrical conductivities via varying dopants and doping concentrations, were configured to chemiresistors for sensing various analytes, including water vapor, NH3, and NO2. Secondary doping with Cl(-) and CSA differentially affected sensing behaviors by having distinctive optimal sensitivities. Biphasic sensitivity with respect to electrical conductivity was observed, demonstrating a facile method to enhance gas sensitivity by optimizing secondary doping. A balance between Debye length of the nanofibers and overall charge conduction may play an important role for modulating such an optimal sensitivity.

18.
Am J Med Genet A ; 161A(8): 2056-9, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23824731

RESUMO

A mother and daughter with an overlapping Catel-Manzke and Temtamy preaxial brachydactyly hyperphalangism syndrome phenotype are reported. We describe a phenotype with a previously undescribed genetic basis. .


Assuntos
Deformidades Congênitas do Pé/diagnóstico , Tórax em Funil/diagnóstico , Deformidades Congênitas da Mão/diagnóstico , Mães , Núcleo Familiar , Hibridização Genômica Comparativa , Feminino , Deformidades Congênitas do Pé/genética , Tórax em Funil/genética , Deformidades Congênitas da Mão/genética , Humanos , Recém-Nascido , Fenótipo
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