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1.
Photodiagnosis Photodyn Ther ; : 101598, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31731066

RESUMO

Hemophilia (HP), the deficiency of clotting factors such as VIII (FVIII) and IX, is an inherited blood disorder which is due to the lack of clotting protein. Conventional techniques for detecting hemophilia are based on clotting factor tests such as hemostasis assays to determine various types of hemophilia and its severity. In the current study, we propose a new approach involving a spectral technique to discriminate normal controls from hemophilia patients with an accuracy above 80%. This accuracy is calculated on the normalized relative intensity based on the light measurement of blood components by analyzing a certain set of fluorescent bio molecules. Our results indicated that Red Blood Cells (RBCs) show lower porphyrin content and enzyme deficiencies in hemophilia patients than in normal controls. Therefore, the spectral features in hemophilia patients are different from those of normal controls. Thus, the proposed technique is a good alternative for the detection of hemophilia patients when compared with conventional detection techniques.

2.
Genet Med ; 21(11): 2532-2542, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31036918

RESUMO

PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.

3.
Am J Hum Genet ; 104(1): 94-111, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609410

RESUMO

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/genética , Linhagem , Síndrome , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
4.
Am J Hum Genet ; 102(5): 744-759, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29656859

RESUMO

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

5.
Am J Hum Genet ; 101(4): 503-515, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28942966

RESUMO

Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes.


Assuntos
Anormalidades Múltiplas/genética , Antígenos Nucleares/genética , Anormalidades Craniofaciais/genética , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência/genética , Transtornos do Desenvolvimento da Linguagem/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/patologia , Adolescente , Animais , Antígenos Nucleares/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Montagem e Desmontagem da Cromatina , Estudos de Coortes , Anormalidades Craniofaciais/patologia , Feminino , Edição de Genes , Haploinsuficiência/fisiologia , Humanos , Transtornos do Desenvolvimento da Linguagem/patologia , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Microcefalia/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
7.
Am J Hum Genet ; 100(4): 666-675, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28318500

RESUMO

Despite the accelerated discovery of genes associated with syndromic traits, the majority of families affected by such conditions remain undiagnosed. Here, we employed whole-exome sequencing in two unrelated consanguineous kindreds with central nervous system (CNS), cardiac, renal, and digit abnormalities. We identified homozygous truncating mutations in TMEM260, a locus predicted to encode numerous splice isoforms. Systematic expression analyses across tissues and developmental stages validated two such isoforms, which differ in the utilization of an internal exon. The mutations in both families map uniquely to the long isoform, raising the possibility of an isoform-specific disorder. Consistent with this notion, RT-PCR of lymphocyte cell lines from one of the kindreds showed reduced levels of only the long isoform, which could be ameliorated by emetine, suggesting that the mutation induces nonsense-mediated decay. Subsequent in vivo testing supported this hypothesis. First, either transient suppression or CRISPR/Cas9 genome editing of zebrafish tmem260 recapitulated key neurological phenotypes. Second, co-injection of morphants with the long human TMEM260 mRNA rescued CNS pathology, whereas the short isoform was significantly less efficient. Finally, immunocytochemical and biochemical studies showed preferential enrichment of the long TMEM260 isoform to the plasma membrane. Together, our data suggest that there is overall reduced, but not ablated, functionality of TMEM260 and that attenuation of the membrane-associated functions of this protein is a principal driver of pathology. These observations contribute to an appreciation of the roles of splice isoforms in genetic disorders and suggest that dissection of the functions of these transcripts will most likely inform pathomechanism.


Assuntos
Anormalidades Múltiplas/genética , Síndrome Cardiorrenal/genética , Proteínas de Membrana/genética , Transtornos do Neurodesenvolvimento/genética , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Mutação Puntual , Isoformas de Proteínas/genética
8.
Am J Hum Genet ; 100(2): 352-363, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28132691

RESUMO

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.


Assuntos
Transtornos do Neurodesenvolvimento/genética , Complexo de Endopeptidases do Proteassoma/genética , Adolescente , Animais , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Deleção de Genes , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Polimorfismo de Nucleotídeo Único , Peixe-Zebra/genética
9.
Hum Mol Genet ; 22(25): 5199-214, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23918663

RESUMO

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.


Assuntos
Centrossomo/metabolismo , Quinase 6 Dependente de Ciclina/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mitose/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 7/genética , Quinase 6 Dependente de Ciclina/química , Quinase 6 Dependente de Ciclina/metabolismo , Feminino , Estudos de Associação Genética , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Microcefalia/fisiopatologia , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Conformação Proteica
10.
Nephrol Dial Transplant ; 19(4): 844-51, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15031339

RESUMO

BACKGROUND: The molecular basis for the alteration of glomerular podocyte phenotype in nephrotic syndrome probably involves adaptive changes of the actin cytoskeleton. alpha-Actinin-4 is an actin cross-linking protein that also interacts with intra- and intercellular adhesion molecules and elements of the transmembrane signal transduction pathway and is implicated in nephrotic syndrome by animal models and human genetic studies. METHODS: We have performed the first quantitative immunoelectron microscopy study of alpha-actinin-4 expression in humans, analysing 12 cases of minimal change nephrosis (MCNS) and 16 cases of idiopathic membranous nephropathy (MGN), and comparing this with expression in normal tissue from seven nephrectomies (Nx). alpha-Actinin-4 was visualized by immunogold labelling of plastic-embedded whole glomerular cross-sections, and analysed using LUCIA software. RESULTS: Despite podocyte effacement, alpha-actinin-4 expression (group mean+/-1 SD) in MCNS was similar to that seen in normal Nx podocytes. In contrast, alpha-actinin-4 expression in MGN was significantly higher than in MCNS or Nx (P<0.001). Furthermore, in MGN cases showing a segmental deposition, expression of alpha-actinin-4 was significantly higher only in those capillary loop segments containing deposits, whereas in those segments without deposits expression was unchanged compared with that seen in Nx (P<0.001). alpha-Actinin-4 expression was higher in MGN cases where subepithelial deposits abutted podocyte cytoplasm, and lower in disease stages where deposits were contained within the glomerular basement membrane or were being reabsorbed. CONCLUSIONS: Elevated alpha-actinin-4 expression is observed only in MGN, and only in areas of subepithelial deposits. Further investigation into the cause of this may reveal insights into the pathogenesis of acquired nephrosis.


Assuntos
Actinina/biossíntese , Proteínas dos Microfilamentos/biossíntese , Síndrome Nefrótica/metabolismo , Actinina/análise , Adolescente , Adulto , Idoso , Criança , Feminino , Ouro , Humanos , Masculino , Proteínas dos Microfilamentos/análise , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Síndrome Nefrótica/patologia
11.
J Histochem Cytochem ; 52(1): 141-4, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14688225

RESUMO

In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.


Assuntos
Actinina/metabolismo , Imuno-Histoquímica/métodos , Integrina beta1/metabolismo , Rim/metabolismo , Proteínas dos Microfilamentos/metabolismo , Crioultramicrotomia , Epitopos , Humanos , Rim/ultraestrutura , Coloração pela Prata
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