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1.
J Cancer Res Clin Oncol ; 145(12): 2891-2899, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617076

RESUMO

PURPOSE: Microsatellites are widely distributed repetitive DNA motifs, accounting for approximately 3% of the genome. Due to mismatch repair system deficiency, insertion or deletion of repetitive units often occurs, leading to microsatellite instability. In this review, we aimed to explore the relationship between MSI and biological behaviour of colorectal carcinoma, gastric carcinoma, lymphoma/leukaemia and endometrial carcinoma, as well as the application of frameshift peptide vaccines in cancer therapy. METHODS: The relevant literature from PubMed and Baidu Xueshu were reviewed in this article. The ClinicalTrials.gov database was searched for clinical trials related to the specific topic. RESULTS: Microsatellite instability is divided into three subtypes: high-level, low-level microsatellite instability, and stable microsatellites. The majority of tumour patients with high-level microsatellite instability often show a better efficacy and prognosis than those with low-level microsatellite instability or stable microsatellites. In coding regions, especially for genes involved in tumourigenesis, microsatellite instability often results in inactivation of proteins and contributes to tumourigenesis. Moreover, the occurrence of microsatellite instability in coding regions can also cause the generation of frameshift peptides that are thought to be unknown and novel to the individual immune system. Thus, these frameshift peptides have the potential to be biomarkers to raise tumour-specific immune responses. CONCLUSION: MSI has the potential to become a key predictor for evaluating the degree of malignancy, efficacy and prognosis of tumours. Clinically, MSI patterns will provide more valuable information for clinicians to create optimal individualized treatment strategies based on frameshift peptides vaccines.


Assuntos
Mutação da Fase de Leitura/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Animais , Carcinogênese/genética , Ensaios Clínicos como Assunto , Reparo de Erro de Pareamento de DNA/genética , Humanos , Instabilidade de Microssatélites , Prognóstico
3.
Biochem Mol Biol Educ ; 47(5): 573-580, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31225941

RESUMO

The CRISPR/Cas9 system is a powerful tool for gene editing and it has become increasingly important for biology students to understand this emerging technique. Most CRISPR laboratory teaching modules use complex metazoan systems or mammalian cell culture which can be expensive. Here, we present a lab module that engages students in learning the fundamentals of CRISPR/Cas9 methodology using the simple and inexpensive model system, Saccharomyces cerevisiae. Students use CRISPR/Cas9 and nonhomologous end joining to generate frameshift insertion and deletion mutations in the CAN1 gene, which are easily selected for using media plates that have canavanine. DNA sequencing is also performed to determine what type of mutation occurred in gene-edited cells. This easy to implement set of experiments has been run as both a 5-week and a shorter 3-week lab module. Learning assessments demonstrate increased understanding in CRISPR-related concepts as well as increased confidence using molecular techniques. Thus, this CRISPR/Cas9 lab module can be added to an existing Genetics, Microbiology, or Molecular Biology lab course to help undergraduate students learn current gene editing techniques with limited effort and cost. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):573-580, 2019.


Assuntos
Sistemas CRISPR-Cas/genética , Mutação da Fase de Leitura/genética , Laboratórios , Saccharomyces cerevisiae/genética , Humanos , Aprendizagem , Estudantes , Universidades
4.
PLoS Genet ; 15(5): e1008130, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048900

RESUMO

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.


Assuntos
Glaucoma de Ângulo Fechado/genética , Hiperopia/genética , Proteínas de Membrana/genética , Microftalmia/genética , Degeneração Retiniana/genética , Fatores de Transcrição/genética , Adulto , Animais , Criança , Pré-Escolar , Éxons , Família , Feminino , Mutação da Fase de Leitura/genética , Variação Genética/genética , Glaucoma de Ângulo Fechado/metabolismo , Humanos , Hiperopia/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/metabolismo , Pessoa de Meia-Idade , Linhagem , Sítios de Splice de RNA/genética , Erros de Refração/genética , Fatores de Transcrição/metabolismo
5.
Genet Test Mol Biomarkers ; 23(6): 423-427, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31063410

RESUMO

Aim: The aim of this study was to report a novel POU Class 3 Homeobox 4 (POU3F4) variant and to provide further guidance on genetic counseling for incomplete partition (IP) type III families in the Korean population by showing two new contrasting cases in terms of genotypes and inheritance. Materials and Methods: Two consecutively recruited hearing-impaired probands with seemingly nonsyndromic features and their biological mothers were included in this study. Sanger sequencing and quantitative polymerase chain reaction (PCR) assays were performed for POU3F4. Results: A novel frameshift variant of POU3F4, c.852delC (p.Ile285Serfs*3), was identified in one of the patients. This mutation is predicted to truncate the protein within the POU homeodomain, resulting in the complete loss of the last nucleus localization signal. The proband's biological mother was also shown to be a carrier of this c.852delC (p.Ile285Serfs*3) mutant allele. A de novo genomic deletion on chromosome Xq21.2 was confirmed in another subject via quantitative PCR. This subject's biological mother, however, was not a carrier of this deletion. This indicates that the large upstream deletion of POU3F4 in the second proband occurred de novo. This finding is compatible with the previously proposed tendency for a high de novo rate of large genomic deletions involving the X-linked deafness-2 (DFNX2) locus. Conclusion: This study adds a novel, probably pathogenic POU3F4 truncation variant to the literature and provides guidance toward effective genetic counseling for IP III subjects based on more frequent de novo occurrence of POU3F4 deletions than POU3F4 point variants.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Perda Auditiva Neurossensorial/genética , Fatores do Domínio POU/genética , Adulto , Criança , Surdez/genética , Família , Feminino , Mutação da Fase de Leitura/genética , Aconselhamento Genético/métodos , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Genótipo , Perda Auditiva/genética , Perda Auditiva Neurossensorial/metabolismo , Humanos , Masculino , Mutação , Linhagem , República da Coreia
6.
J Med Case Rep ; 13(1): 101, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31014398

RESUMO

BACKGROUND: Hypophosphatasia is an inherited bone disease characterized by low alkaline phosphatase activity encoded by ALPL. Clinically, hypophosphatasia can be categorized as perinatal, infantile, childhood, and adult forms, as well as odonto-hypophosphatasia, according to the age at first sign or dental manifestations. Adult hypophosphatasia typically presents in middle-aged patients who appear to be in good health in early adulthood and manifests as painful feet caused by recurrent, slow-healing stress fractures of the lower limb. Because the symptoms of adult hypophosphatasia vary and are common, many patients with hypophosphatasia might be not diagnosed accurately and thus may receive inappropriate treatment. CASE PRESENTATION: We report a case of a 35-year-old Japanese woman with low serum alkaline phosphatase detected at a routine medical checkup. She had mild muscle/bone pain but no history of rickets, fractures, or dental problems. Measurement of bone mineral density of the lumbar spine and the femoral neck revealed osteopenia below the expected range for age in a young adult. Abdominal ultrasonography revealed numerous microcalcifications in both kidneys. Analysis of amino acids in urine revealed that phosphoethanolamine was elevated. Low serum alkaline phosphatase activity, elevation of phosphoethanolamine, and low bone mineral density supported the diagnosis of hypophosphatasia. ALPL mutation analysis revealed two mutations: p.Phe327Leu and c.1559delT. These genetic abnormalities were previously reported in perinatal, infantile, and childhood but not adult hypophosphatasia. On the basis of the clinical presentation, laboratory and imaging findings, and genetic analyses, the patient was definitively diagnosed with adult hypophosphatasia. To the best of our knowledge, this is the first case report of adult hypophosphatasia with the compound heterozygous mutations p.Phe327Leu and c.1559delT. CONCLUSIONS: Although the risk of bone fracture was high in this case, treatment approaches differ between osteoporosis and hypophosphatasia. Because adult hypophosphatasia diagnosis is often difficult because of their varied symptoms, hypophosphatasia should be considered in the differential diagnosis of low serum alkaline phosphatase. Early diagnosis is important so that appropriate treatment can be initiated.


Assuntos
Fosfatase Alcalina/sangue , Fraturas Espontâneas/genética , Mutação da Fase de Leitura/genética , Hipofosfatasia/genética , Adulto , Análise Mutacional de DNA , Feminino , Fraturas Espontâneas/sangue , Fraturas Espontâneas/fisiopatologia , Humanos , Hipofosfatasia/sangue , Hipofosfatasia/complicações , Hipofosfatasia/fisiopatologia , Mutação de Sentido Incorreto
7.
Hum Genet ; 138(5): 509-513, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847549

RESUMO

Startle disease, or hyperekplexia, is a glycinergic disorder characterized by hypertonia and apnea that is triggered by noise and/or touch. Mutations in five genes have been associated with startle disease in humans, dogs, cattle, and mice. We identified a novel recessive startle disease in a family of Spanish greyhounds. Whole genome resequencing of an affected dog revealed a homozygous two base pair deletion in the ninth exon of SLC6A5, encoding the presynaptic glycine transporter. The deletion is predicted to cause a frameshift, p.S460FfsX47, leading to a premature stop codon that truncates over a third of the protein. Family members were genotyped for the deletion, and findings were consistent with an autosomal recessive inheritance pattern. The pathogenic variant was absent from 34 unrelated greyhounds, 659 domestic dogs of pure and mixed breeds, and 54 wild canids, suggesting it occurred recently and may be private to the family. The findings of this study can be used to inform future breeding decisions and prevent dissemination of the deleterious allele in greyhounds.


Assuntos
Doenças do Cão/genética , Mutação da Fase de Leitura/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Rigidez Muscular Espasmódica/genética , Rigidez Muscular Espasmódica/veterinária , Animais , Códon sem Sentido/genética , Modelos Animais de Doenças , Cães , Deleção de Sequência/genética , Sequenciamento Completo do Genoma
8.
Hum Genet ; 138(5): 525-533, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877375

RESUMO

Amelogenesis imperfecta (AI) refers to a genetically and clinically heterogeneous group of inherited disorders affecting the structure, composition, and quantity of tooth enamel. Both non-syndromic and syndromic forms of AI have been described and several genes affecting various aspects of the enamel physiology have been reported. Genetically modified murine models of various genes have provided insights into the complex regulation of proper amelogenesis. Non-syndromic AI occurs spontaneously also in dogs with known recessive variants in ENAM and SLC24A4 genes. Unlike rodents with a reduced dentition and continuously erupting incisors, canine models are valuable for human AI due to similarity in the dental anatomy including deciduous and permanent teeth. We have performed a series of clinical and genetic analyses to investigate AI in several breeds of dogs and describe here two novel recessive variants in the ENAM and ACP4 genes. A fully segregating missense variant (c.716C>T) in exon 8 of ENAM substitutes a well-conserved proline to leucine, p.(Pro239Leu), resulting in a clinical hypomineralization of teeth. A 1-bp insertion in ACP4 (c.1189dupG) is predicted to lead to a frameshift, p.(Ala397Glyfs), resulting in an abnormal C-terminal part of the protein, and hypoplastic AI. The ENAM variant was specific for Parson Russell Terriers with a carrier frequency of 9%. The ACP4 variant was found in two breeds, Akita and American Akita with a carrier frequency of 22%. These genetic findings establish novel canine models of human AI with a particular interest in the case of the ACP4-deficient model, since ACP4 physiology is poorly characterized in human AI. The affected dogs could also serve as preclinical models for novel treatments while the breeds would benefit from genetic tests devised here for veterinary diagnostics and breeding programs.


Assuntos
Amelogênese Imperfeita/genética , Amelogênese Imperfeita/veterinária , Proteínas do Esmalte Dentário/genética , Esmalte Dentário/fisiopatologia , Fosfatase Ácida Resistente a Tartarato/genética , Animais , Modelos Animais de Doenças , Cães , Mutação da Fase de Leitura/genética , Genótipo , Humanos , Mutação de Sentido Incorreto/genética
9.
Hum Genet ; 138(5): 535-539, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30927068

RESUMO

Hairlessness is a breed-specific feature selected for in some dog breeds but a rare abnormality in some others such as Scottish Deerhounds (SD). In SDs, the affected puppies are born with sparse hair but lose it within the first 2 months leaving the dogs completely hairless. The previous studies have implicated variants in FOXI3 and SGK3 in hairlessness; however, the known variants do not explain hairlessness in all breeds such as SDs. We investigated the genetic cause in 66 SDs, including a litter with two hairless dogs. We utilized a combined approach of genome-wide homozygosity mapping and whole-genome sequencing of a hairless SD followed by recessive filtering according to a recessive model against 340 control genomes. Only two homozygous-coding variants were discovered in the homozygosity regions, including a 1-bp insertion in exon 2 of SGK3. This results in a predicted frameshift and very early truncation (49/490 amino acids) of the SGK3 protein. Additional screening of the recessive variant demonstrated a full segregation with the hairlessness and a 12% carrier frequency in the SD breed. The variant was not found in the related Irish Wolfhound breed. This study identifies the second hairless variant in the SGK3 gene in dogs and further highlights its role as a candidate gene for androgen-independent hair loss or alopecia in human.


Assuntos
Alopecia/genética , Alopecia/veterinária , Doenças do Cão/genética , Proteínas Imediatamente Precoces/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Modelos Animais de Doenças , Cães , Feminino , Mutação da Fase de Leitura/genética , Humanos , Masculino , Sequenciamento Completo do Genoma
10.
Brain Dev ; 41(6): 538-541, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30795918

RESUMO

PPM1D truncating mutations in the last and penultimate exons of the gene have been associated with intellectual disability (ID) syndrome. Only 15 affected patients to-date have been reported with mild-to-severe ID, autistic behavior, anxiety and dysmorphic features. Here, we describe the clinical characteristics and underlying genetics of two unrelated girls with moderate developmental delay and dysmorphic features associated with novel mutations in PPM1D exon 5. The dysmorphic features demonstrated by these two patients are consistent with previously reported patients, including broad forehead, thin upper lip, brachydactyly, and hypoplastic nails. We identified a de novo PPM1D mutation in exon 5 of each patient (c.1250_1251insACCA p.V419Tfs*16 and c.1256_1257insCAAG p.S421Qfs*14) by panel sequencing for 4,813 disease-related genes. Both patients also had frameshift mutations (at different positions) that resulted in the same estimated termination codon at 434. These additional reports add to the growing literature on PPM1D-associated ID syndrome and help delineate the clinical phenotype and genetic basis.


Assuntos
Deficiência Intelectual/genética , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/fisiologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Éxons/genética , Feminino , Mutação da Fase de Leitura/genética , Genótipo , Humanos , Deficiência Intelectual/metabolismo , Mutação/genética , Fenótipo , Sequenciamento Completo do Exoma/métodos
11.
Iran Biomed J ; 23(3): 228-34, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30797226

RESUMO

Background: Long QT syndrome (LQTS) is characterized by the prolongation of QT interval, which results in syncope and sudden cardiac death in young people. KCNQ1 is the most common gene responsible for this syndrome. Methods: Molecular investigation was performed by DNA Sanger sequencing in Iranian families with a history of syncope. In silico examinations were performed for predicting the pathogenicity of the novel variant. Results: A novel homozygous KCNQ1 frameshift mutation, c.1426_1429delATGC (M476Pfs*4), was identified, and then the current literatures of five patients were reviewed regarding the LQTS. Conclusion: The novel frameshift mutation has been reported for the first time among the Iranian population. Our finding along with the case series study of LQTS patients illustrates the importance of genetic and case series in precise detection of the frequency of LQTS carriers.


Assuntos
Mutação da Fase de Leitura/genética , Predisposição Genética para Doença , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Sequência de Bases , Eletrocardiografia , Feminino , Humanos , Irã (Geográfico) , Síndrome do QT Longo/diagnóstico por imagem , Masculino , Linhagem
12.
Cancer Genet ; 231-232: 67-79, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30803560

RESUMO

BACKGROUND: Usually, genes with a higher-than-expected number of somatic mutations in tumor samples are assumed to be cancer related. We identified genes with a fewer-than-expected number of somatic mutations - "untouchable genes". METHODS: To predict the expected number of somatic mutations, we used a linear regression model with the number of mutations in the gene as an outcome, and gene characteristics, including gene size, nucleotide composition, level of evolutionary conservation, expression level and others, as predictors. Analysis of residuals from the regression model was used to compare the observed and predicted number of mutations. RESULTS: We have identified 19 genes with a less-than-expected number of loss-off-function (nonsense, frameshift or pathogenic missense) mutations - i.e., untouchable genes. The number of silent or neutral missense mutations in untouchable genes was equal or higher than the expected number. Many mucins, including MUC16, MUC17, MUC6, MUC5AC, MUC5B, and MUC12, are untouchable. We hypothesized that untouchable mucins help tumor cells to avoid immune response by providing a protective coat that prevents direct contact between effector immune cells, e.g., cytotoxic T-cells, and tumor cells. Survival analysis of available TCGA data demonstrated that overall survival of patients with low (below the median) expression of untouchable mucins was better compared to patients with high expression of untouchable mucins. Aside from mucins, we have identified a number of other untouchable genes. CONCLUSIONS: Untouchable genes may be ideal targets for cancer treatment since suppression of untouchable genes is expected to inhibit survival of tumor cells.


Assuntos
Genes Neoplásicos , Genoma Humano , Neoplasias/genética , Neoplasias/terapia , Códon sem Sentido/genética , Mutação da Fase de Leitura/genética , Humanos , Modelos Lineares , Mutação com Perda de Função/genética , Mucinas/genética , Mutação de Sentido Incorreto/genética , Análise de Sobrevida
13.
BMC Vet Res ; 15(1): 62, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777056

RESUMO

BACKGROUND: Vitamin D-dependent rickets is rare in animals and humans. Several types of this condition are associated with genetic variants related to vitamin D metabolism. This is the first report of type 1B vitamin D-dependent rickets in a cat. CASE PRESENTATION: Here, we describe the case of a 3-month-old female domestic short-haired cat previously fed on commercial kitten food that presented at our clinic with seizures, lethargy, and generalized pain. Serum and ionized calcium concentrations and 1,25-dihydroxycholecalciferol in this cat were low, and radiographs showed skeletal demineralization and abnormally wide growth plates on the long bones. Initially, simple vitamin D deficiency was suspected; however, the cat's profile, which included fed a well-balanced commercial diet, together with the findings of additional laboratory tests and the cat's unresponsiveness to various treatments, raised the suspicion of vitamin D-dependent rickets. Examination of the DNA sequences of CYP2R1 and CYP27B1 genes, which are genes linked with vitamin D metabolism, showed a CYP2R1 frameshift mutation in exon 5 (where T is deleted at position c.1386). This mutation alters the amino acid sequence from position 462, while the stop codon introduced at position 481 prematurely truncates the 501 amino acid full-length protein. With this knowledge, a new treatment regime based on a standard dose of calcitriol was started and this markedly improved the cat's condition. CONCLUSIONS: To the best of our knowledge, the present case is the first description of type 1B vitamin D-dependent rickets linked with a genetic variant of CYP2R1 in a cat.


Assuntos
Doenças do Gato/genética , Família 2 do Citocromo P450/genética , Raquitismo Hipofosfatêmico Familiar/veterinária , Animais , Calcitriol/uso terapêutico , Doenças do Gato/diagnóstico , Doenças do Gato/tratamento farmacológico , Gatos , Raquitismo Hipofosfatêmico Familiar/diagnóstico , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Raquitismo Hipofosfatêmico Familiar/genética , Feminino , Mutação da Fase de Leitura/genética , Análise de Sequência de DNA/veterinária , Vitaminas/uso terapêutico
14.
Genet Test Mol Biomarkers ; 23(3): 204-208, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30758234

RESUMO

AIMS: Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of hereditary deafness. Despite its frequency, the diagnosis of this disorder continues to be a challenging task given its extreme genetic heterogeneity. The purpose of this study was to identify the causative mutation in a consanguineous United Arab Emirates (UAE) family with ARNSHL. MATERIALS AND METHODS: Clinical exome sequencing (CES) followed by segregation analysis via Sanger sequencing was used to identify the causative mutation. In addition, 109 deaf individuals and 50 deafness-free controls from the UAE population were screened for the identified mutation. RESULTS AND DISCUSSION: CES identified the STRC frameshift mutation c.4510del (p.Glu1504Argfs*32) as the causative mutation in this family. Moreover, segregation analysis confirmed the above finding. In addition, the absence of this variant in 109 unrelated deaf individuals and 50 healthy controls indicates that it is rare in the UAE population. CONCLUSION: The present study represents the first STRC mutation reported in the UAE population. It also reinforces the power of next-generation sequencing in the diagnosis of heterogenous disorders such as nonsyndromic hearing loss.


Assuntos
Surdez/genética , Proteínas de Membrana/genética , Adulto , Exoma/genética , Feminino , Mutação da Fase de Leitura/genética , Genes Recessivos/genética , Perda Auditiva Neurossensorial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Proteínas de Membrana/fisiologia , Mutação , Linhagem , Emirados Árabes Unidos , Sequenciamento Completo do Exoma
15.
Orphanet J Rare Dis ; 14(1): 45, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770747

RESUMO

BACKGROUND: Cornelia de Lange syndrome (CdLS) and Rubinstein-Taybi syndrome (RSTS) are both rare congenital multiple malformation disorders caused by genes associated with transcription. They share a number of similar features clinically. In addition, it is difficult to make a molecular diagnosis rapidly and detect the mosaic mutation when only sanger sequencing is taken. This study aims to report three novel mutations in three Chinese children identified by next generation sequencing. RESULTS: We describe patient 1 and patient 2 presenting with characteristics of CdLS with mutations in NIPBL and patient 3 with a frame shift mutation in CREBBP who can be diagnosed as RSTS clinically and also have similar symptoms with CdLS to some extent. The splicing site c.4321-1G > A transversion in NIPBL is a mosaic mutation and produces an abnormal transcript bearing the loss of exon 20. The nonsense mutation c.218C > A in NIPBL and the frame shift c.1715delC mutation in CREBBP generate stop codon and yield the premature termination of proteins. CONCLUSIONS: In general, we detect three novel heterozygous mutations including a splicing mutation and a nonsense mutation in NIPBL and a frame shift in CREBBP. And several similar features observed in patients indicate the clinical complexity and clinically overlapping of CdLS and RSTS termed "transcriptomopathies", suggest the underlying molecular mechanism and emphasize the utilization of next generation sequencing technologies.


Assuntos
Proteína de Ligação a CREB/genética , Mutação da Fase de Leitura/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Proteínas/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Masculino , Síndrome de Rubinstein-Taybi/genética
16.
Methods Mol Biol ; 1940: 77-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788819

RESUMO

Efficient and reliable methods for gene editing are critical for the generation of loss-of-gene function stem cells and genetically modified mice. Here, we outline the application of CRISPR-Cas9 technology for gene editing in mouse embryonic stem cells (mESCs) to generate knockout ESC chimeras for the fast-tracked analysis of gene function. Furthermore, we describe the application of gene editing directly to mouse epiblast stem cells (mEpiSCs) for modelling germ layer differentiation in vitro.


Assuntos
Mutação da Fase de Leitura/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Camundongos , Plasmídeos/genética , RNA Guia/genética
17.
Medicine (Baltimore) ; 97(50): e13565, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30558019

RESUMO

Dravet syndrome is considered to be one of the most severe types of genetic epilepsy. Mutations in SCN1A gene have been found to be responsible for at least 80% of patients with Dravet syndrome, and 90% of these mutations arise de novo. The variable clinical phenotype is commonly observed among these patients with SCN1A mutations, suggesting that genetic modifiers may influence the phenotypic expression of Dravet syndrome. In the present study, we described the clinical, pathological, and molecular characteristics of 13 Han Chinese pedigrees clinically diagnosed with Dravet syndrome. By targeted-exome sequencing, bioinformatics analysis and Sanger sequencing verification, 11 variants were identified in SCN1A gene among 11 pedigrees including 7 missense mutations, 2 splice site mutations, and 2 frameshift mutations (9 novel variants and 2 reported mutations). Particularly, 2 of these Dravet syndrome patients with SCN1A variants also harbored SCN9A, KCNQ2, or SLC6A8 variants. In addition, 2 subjects were failed to detect any pathogenic mutations in SCN1A and other epilepsy-related genes. These data suggested that SCN1A variants account for about 84.6% of Dravet syndrome in our cohort. This study expanded the mutational spectrum for the SCN1A gene, and also provided clinical and genetic evidence for the hypothesis that genetic modifiers may contribute to the variable manifestation of Dravet syndrome patients with SCN1A mutations. Thus, targeted-exome sequencing will make it possible to detect the interactions of epilepsy-related genes and reveal their modification on the severity of SCN1A mutation-related Dravet syndrome.


Assuntos
Epilepsias Mioclônicas/genética , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Linhagem , Análise de Sequência/métodos , Grupo com Ancestrais do Continente Asiático/genética , Pré-Escolar , China , Feminino , Mutação da Fase de Leitura/genética , Humanos , Lactente , Canal de Potássio KCNQ2/genética , Masculino , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Proteínas do Tecido Nervoso/genética , Fenótipo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Isoformas de Proteínas/genética
18.
Mol Vis ; 24: 587-602, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30210230

RESUMO

Purpose: Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1. Methods: One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes. Results: Using gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane. Conclusions: The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.


Assuntos
Modelos Animais de Doenças , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/patologia , Rodopsina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Códon de Terminação/genética , Mutação da Fase de Leitura/genética , Marcação de Genes , Immunoblotting , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Degeneração Retiniana/patologia
19.
Medicine (Baltimore) ; 97(33): e11636, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30113454

RESUMO

INTRODUCTION: Cockayne syndrome (CS) is a rare multisystemic autosomal recessive disease. The primary manifestations of which are developmental delay, neurological impairment, abnormal skin sensitivity to sunlight and unique facial appearance as sunken eyes, large ears, and thin large nose. The disorders of the nucleotide excision repair system significantly are caused by mutations of Excision repair cross-complementing group 6 (ERCC6) and Excision repair cross-complementing group 8 (ERCC8) genes, and the ERCC6 gene mutations are present in approximately 65% of cases. CASE PRESENTATION: Here we described a girl in a consanguineous Jordanian family with abnormal facial appearance and postnatal growth delay. She was not able to gain weight. Her condition deteriorated progressively and she developed difficulty of swallowing even to water. The patient was diagnosed as CS based on her facial appearance and neurologic dysfunction. The patient was examined at 3 years old, and died at 4 years old. CONCLUSION: Genetic analysis and sequencing revealed homozygosity for a novel frame shift mutation c.2911_2915del5ins9 (p.Lys971TryfsX14) in the ERCC6. The mutation is predicted to delete 5 nucleotides and add 9 nucleotides with a premature termination, resulting in approximately 34% length reduction of the wild-type transcript. The multisystem malformations of CS are clinically heterogeneous. The frame shift mutation of ERCC6 found in this patient is a novel one, which caused postnatal growth failure and early death. Our findings indicate truncated mutation in CS lead to more severe CS phenotype and add to the genotype-phenotype correlations in CS.


Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Deficiências do Desenvolvimento/diagnóstico , Mutação da Fase de Leitura/genética , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Pré-Escolar , Síndrome de Cockayne/complicações , Síndrome de Cockayne/mortalidade , Consanguinidade , Reparo do DNA/genética , Deficiências do Desenvolvimento/etiologia , Evolução Fatal , Feminino , Estudos de Associação Genética/métodos , Transtornos do Crescimento/genética , Crescimento e Desenvolvimento/genética , Humanos , Jordânia/epidemiologia , Fenótipo
20.
Medicine (Baltimore) ; 97(32): e11499, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30095615

RESUMO

BACKGROUND: Cone-rod dystrophy (CORD) is an inherited, progressive retinal disorder with genetic and phenotypic heterogeneity. Here, we aimed to identify the pathogenic mutation in affected individuals in a Chinese family with autosomal dominant cone-rod dystrophy (adCORD). METHODS: Genomic DNA and clinical examination results were collected from a Chinese family presenting with adCORD. The candidate disease-causing mutations were screened with whole-exome sequencing (WES) and bioinformatics analyses. Sanger sequencing was used for validation and cosegregation analysis. RESULTS: A novel frameshift mutation (NM_000554.4; c.538dupG:p.Val180fs) in exon 4 of the CRX gene was identified in all affected individuals in the Chinese family with adCORD. Cosegregation analysis confirmed that this mutation was cosegregated with the disease. This variant, which results in premature termination of the protein, was absent from all public variant databases or internal exome databases. CONCLUSIONS: We used whole-exome sequencing to identify a novel CRX mutation causing adCORD in a Chinese family. This study broadens the known pathogenic mutation spectrum of the CRX gene and shows the potential of WES in identifying the pathogenic mutations of CORD disease.


Assuntos
Distrofias de Cones e Bastonetes/genética , Mutação da Fase de Leitura/genética , Proteínas de Homeodomínio/genética , Transativadores/genética , Adolescente , Grupo com Ancestrais do Continente Asiático/genética , China , Feminino , Humanos , Masculino , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
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