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The concept of benefit finding, the assessment tools and the status quo of benefit finding for family caregivers of stroke patients were elaborated, the influencing factors of benefit finding of family caregivers of stroke patients were summarized, the current problems and the development direction of future research were pointed out, aiming to provide a reference for clinical staff to conduct research on benefit finding of family caregivers of stroke patients in China.
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Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.
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Objective@#To develop a system for rapid detection of JAK2 V617F mutation among patients with myeloproliferative diseases.@*Methods@#Specific primers and TagMan probes were designed for the mutant and wild type alleles based on the principle of real-time PCR. A complete system including the method for detection and product for quality control were established through the evaluation of sensitivity and accuracy of the method, double-blind trial, and preparation of negative and positive controls through site-directed mutagenesis and molecular cloning.@*Results@#A system for rapid detection of the JAK V617F mutation has been developed. Compared with Sanger sequencing, the sensitivity and specificity of the method have both reached 100%. Meanwhile, 1000 normal samples and 1 case with the JAK2 V617F mutation were detected, which gave a population rate of 1‰.@*Conclusion@#The system was fast, accurate, cheap, high throughput, and easy to use. It can be utilized as a routine test. Although the JAK2 V617F mutation is rare in the population, it should be screened among myeloproliferative neoplasm patients.
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OBJECTIVE@#To develop a system for rapid detection of JAK2 V617F mutation among patients with myeloproliferative diseases.@*METHODS@#Specific primers and TagMan probes were designed for the mutant and wild type alleles based on the principle of real-time PCR. A complete system including the method for detection and product for quality control were established through the evaluation of sensitivity and accuracy of the method, double-blind trial, and preparation of negative and positive controls through site-directed mutagenesis and molecular cloning.@*RESULTS@#A system for rapid detection of the JAK V617F mutation has been developed. Compared with Sanger sequencing, the sensitivity and specificity of the method have both reached 100%. Meanwhile, 1000 normal samples and 1 case with the JAK2 V617F mutation were detected, which gave a population rate of 1‰.@*CONCLUSION@#The system was fast, accurate, cheap, high throughput, and easy to use. It can be utilized as a routine test. Although the JAK2 V617F mutation is rare in the population, it should be screened among myeloproliferative neoplasm patients.
Subject(s)
Humans , Alleles , DNA Mutational Analysis , Double-Blind Method , Janus Kinase 2 , Genetics , Mutation , Myeloproliferative Disorders , Genetics , Real-Time Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular, and cellular interactions. Ameloblastin (AMBN, also named "amelin" or "sheathlin") is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta (AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders. We recruited one kindred with autosomal-dominant amelogenesis imperfecta (ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Amelogenesis Imperfecta , Genetics , Cells, Cultured , China , Codon , Dentin , Congenital Abnormalities , Microsatellite Repeats , Microscopy, Electron, Scanning , Mutation, Missense , Pedigree , RNA , Transfection , Exome SequencingABSTRACT
<p><b>OBJECTIVE</b>To analyze the clinical phenotype of a Chinese pedigree affected with hereditary dentinogenesis imperfecta and mutation of dentin sialophosphoprotein (DSPP) gene.</p><p><b>METHODS</b>Affected members underwent intraoral photography, dental film and panoramic radiography. Genomic DNA was extracted from peripheral venous blood samples. Coding regions of the DSPP gene were subjected to PCR amplification and Sanger sequencing. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. The tertiary structure of wild type and mutant proteins were predicted by Swiss-Port.</p><p><b>RESULTS</b>A heterozygous c.50C to T (p.P17L) mutation was identified in exon 2 of the DSPP gene in the proband and her father. The same mutation was not found among 200 unrelated healthy controls. The Pro-17 residues and its surrounding positions in DSPP are highly conserved across various species. The mutation was predicted to be damaging to the structure of DSPP protein.</p><p><b>CONCLUSION</b>The c.50C to T (p.P17L) mutation of the DSPP gene probably underlies the disease in this pedigree. Above finding has expanded the spectrum of DSPP gene mutations and provided a basis for genetic counseling and prenatal diagnosis for this family.</p>
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<p><b>OBJECTIVE</b>To analyze the genotype of a patient suspected for thalassemia through a series of experiments.</p><p><b>METHODS</b>Conventional methods for detecting common thalassemia mutations was used in conjunction with multiplex ligation-dependent probe amplification (MLPA) in order to determine the genotype of the patient. Corresponding primers were designed for developing a Gap-PCR system for detecting rare type mutations.</p><p><b>RESULTS</b>The patient was identified as a homozygote for Chinese Gγ(Aγδβ)-thal deletion, with clinical manifestations tending to be intermediate or severe based on the hematological characteristics. A Gap-PCR system has been developed for detecting the above mutation with accuracy and rapidity.</p><p><b>CONCLUSION</b>The Chinese Gγ(Aγδβ)-thal is prevalent in southern China, and caution should be taken to avoid misdiagnosis. The Gap-PCR system for detecting Chinese Gγ(Aγδβ)-thal is suitable for extended applications for its simplicity and rapidity.</p>
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<p><b>OBJECTIVE</b>To analyze the clinical phenotype of a Chinese pedigree affected with Papillon-Lefevre syndrome(PLS) and detect mutation of CTSC gene.</p><p><b>METHODS</b>Clinical phenotypes were noted, and oral examination for the proband was carried out for the clinical diagnosis of PLS. PCR and Sanger sequencing were used to identify potential mutation of the CTSC gene. Functional effect of the mutation was predicted with SIFT and PolyPhen-2. Swiss-Port was used to predict the tertiary structure of wild type and mutant proteins. The mRNA and protein expression were analyzed by real-time PCR and Western blotting.</p><p><b>RESULTS</b>A homozygous mutation c.901G>A (p.G301S) in exon 7 of CTSC gene was identified in the patient. Both parents of the patient had carried a heterozygous c.901G>A mutation. The mutation was located in the conserved region of CTSC enzyme and was predicted to be damaging by changing the structure of the protein, which could affect the activity of Cathepsin C. However, no significant difference was found in the expression of p.G301S variant at the mRNA and protein levels compared with that of the wild type CTSC gene.</p><p><b>CONCLUSION</b>The c.901G>A mutation of the CTSC gene was first reported in China, which has expanded its mutation spectrum.</p>
Subject(s)
Adult , Child, Preschool , Female , Humans , Male , Asian People , Genetics , Base Sequence , Cathepsin C , Genetics , China , Exons , Molecular Sequence Data , Mutation , Papillon-Lefevre Disease , Genetics , PedigreeABSTRACT
ObjectiveTo report the results of preventive control program of severe thalassemias in Zhuhai City of Guangdong Province from 1998 to 2010.MethodsAs the guide centre of marriage and childbearing and the greatest maternity hospital in Zhuhai City of Guangdong Province,Zhuhai Municipal Maternity and Child Healthcare Hospital constructed the genetic screening network for thalassemias testing and referred for follow-up and for genetic counseling.The couples for premarital medical examination or regular healthcare examination in pregnancy were enrolled to this preventive control program.A conventional strategy of screening for heterozygote was used to identify the α- and β-thalassemia traits in women and their spouses according to the standard procedures of hematological phenotype analysis which was recommended by Thalassemia International Federation (T IF).Then those suspected couples at risk were diagnosed for α- and β-thalassemia by PCR-based DNA assays.The couples at risk for severe thalassemias were counseled and offered prenatal diagnosis and termination of pregnancy in case of an affected fetus in the rights of consent and of option voluntarily.ResultsFrom January 1998 to December 2010,85 522 brides and grooms-to-be for premarital screening and 41 503 pregnant women in addition to 14 141 partners for prenatal screening were recorded,the covering rates of premarital screening and prenatal screening in the city were 92.698％ (from 1998 to 2003) and 27.667％ (from 2004 to 2010),respectively.Totally 10 726 cases were found to be the carriers of thalassemias,with 7393 for o-thalassemia (5.237％,7 393/141 166) and 3333 for β-thalassemia (2.361％,3 333/141 166).A total of 257 couples at-risk for severe thalassemias were detected including 190 for α-thalassemia and 67 for β-thalassemia.Among them,251 (97.7％,251/257) couples were performed prenatal diagnosis.During the preventive control program,a total of 72 fetuses with severe thalassemias including hemoglobin H disease were voluntarily terminated.In Zhuhai City,the average annual birth rate of fetuses with severe thalassemia was declined by 32.9％ (49/149).ConclusionsThis study has reduced effectively birth rate of perinatal infants with severe thalassemias in Zhuhai City by genetic screening and prenatal diagnosis of thalassemia in the large population of 13 years.Our summary comes out of technical proposals for prenatal screening and diagnosis,which could be take example by preventative control of thalassemia in other regions of China where are prevalent.
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<p><b>OBJECTIVE</b>To develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population.</p><p><b>METHODS</b>Four groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation.</p><p><b>RESULTS</b>Homozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families.</p><p><b>CONCLUSION</b>The single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics.</p>
Subject(s)
Female , Humans , Pregnancy , Asian People , Genetics , China , Heterozygote , Homozygote , Polymerase Chain Reaction , Methods , Prenatal Diagnosis , Reproducibility of Results , Sensitivity and Specificity , alpha-Thalassemia , Diagnosis , Ethnology , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM).</p><p><b>METHODS</b>This protocol is based on the design of two different plasmid DNA templates. Template 1 lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. This modification avoids amplification of full-length wild-type sequences while two templates, the forward and reverse flanking primers exist simultaneously in one reaction tube. A megaprimer is synthesized in the first PCR reaction using template 1, forward primer and mutagenic primer. The megaprimer that needn't the cumbersome gel purification step is directly added to the second PCR reaction system. The second PCR reaction(PCR 2) containing two stages is performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer is extended to form the full-length mutation product. In the second stage of PCR 2, the extended megaprimer containing SDM residues is subsequently amplified with the two flanking primers. All of the final PCR products contained the desired mutation.</p><p><b>RESULTS</b>Fifteen types of rare beta-thalassemia mutations in Chinese were obtained using this method. Each of these modified fragments was separately cloned into the pGEM-T vector and sequenced. The desired mutations involved in mutagenesis amplicons were identified in all clones.</p><p><b>CONCLUSION</b>This improved PCR-based megaprimer method for site-directed mutagenesis is rapid, simple and highly efficient, and the success rate of mutagenesis could reach 100%. Furthermore, this method is suitable for routine application in molecular cloning.</p>
Subject(s)
Humans , Asian People , Genetics , DNA Primers , Globins , Genetics , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Methods , beta-Thalassemia , Ethnology , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the gene frequencies and mutation patterns of alpha thalassemia (alpha-thal) and beta thalassemia (beta-thal) in Liuzhou city of Guangxi Zhuang Autonomous Region.</p><p><b>METHODS</b>Cluster sampling was used. A total of 1 028 of umbilical blood samples were collected for a prevalence study of alpha-thal and a total of 1 312 healthy young people when receiving pre-marriage consultation were recruited for a beta-thal prevalence survey. Individuals live in city or town area of Liuzhou. A complete blood count as well as hemoglobin electrophoresis analysis were done in all of samples for phenotyping of alpha and beta-thals. Those with Hb Bart's for alpha-thal indicator and those with both microcytosis (MCV < 85 fl) and elevated levels of Hb A(2) (>/=4.0%) for beta-thal were further studied by DNA analysis. PCR-based methodologies were used to characterize the mutation contributions of alpha and beta-thals. All the subjects were tested for the state of carrying beta-thala alleles for evaluating the situation of the compound heterozygotes of alpha-thal with beta-thal.</p><p><b>RESULTS</b>Of 1 028 random samples of umbilical blood screened, 112 of subjects were defined to be the gene carriers of alpha-thal. The alpha-thal carrier rate was as high as 11.19% including 3 compound heterozygotes. Five well-known types of alpha-thal alleles were detected with gene contributions of 37.4% (--(SEA) deletion), 31.3% (-alpha(3.7) deletion), 17.4% (-alpha(4.2) deletion), 12.1% (alpha(CS)alpha mutation), and 0.9% (alpha(QS)alpha mutation), successively. Of the 1 312 adult specimens studied, 89 with beta-thal including 14 of the compound higher Hb F subjects were detected. All of the 89 phenotypic beta-thal carriers had the mutations in the beta-globin gene, making the overall prevalence 6.78%. The commonly seen three mutations, beta CD41 - 42 (-CTTT) frameshift, beta CD17 (T-A) nonsense mutation and beta-28 (A-G) promoter variation were accounted for 90% of the beta-thal alleles in Liuzhou. Of these beta-thal subjects, 16 (accounting for 18%) were found to be the compound heterozygosity for a beta-thal and an alpha-thal with 9 different types of gene defects with a detection rate 1.22%.</p><p><b>CONCLUSION</b>Data from ecidation of alpha and beta-thal gene frequencies and mutation spectrum in Liuzhou city was useful for genetic counselling and prenatal diagnosis of this disease.</p>