RESUMO
OBJECTIVE: To screen for potential mutations of KIT gene for two Chinese families affected with piebaldism in order to facilitate genetic counseling and assisted reproduction. METHODS: Peripheral blood samples were collected from 2 patients of family 1 and the proband and 3 unaffected members of family 2 for the extraction of DNA and RNA. PCR-sequencing and reverse transcription PCR-sequencing were used to screen KIT mutations. RESULTS: All of the patients from family 1 were found to carry heterozygous IVS12+2-+7delinsACATCTTTA, a splicing mutation undocumented in the human gene mutation data base (HGMD) database. This mutation has resulted in c.1765-1779del in cDNA and p.Gly592Ala/del:E12, which has led to skipping of exon 12 and no expression of cDNA. The proband from family 2 has carried a heterozygous c.2401A>C mutation in KIT gene. The same mutation was not found in unaffected members. CONCLUSION: We have attained definite diagnosis for both families, which has facilitated genetic counseling and assisted reproduction for our patients and their family members.
Assuntos
Povo Asiático/genética , Mutação da Fase de Leitura , Piebaldismo/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Sequência de Bases , Criança , China , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Adulto JovemRESUMO
OBJECTIVE: To determine the karyotype of a boy suspected to have Cri du Chat syndrome with severe clinical manifestations, and to assess the recurrence risk for his family. METHODS: High-resolution GTG banding was performed to analyze the patient and his parents. Fluorescence in situ hybridization (FISH) with Cri du Chat syndrome region probe as well as subregional probes mapped to 5pter, 5qter, 18pter, 18qter, and whole chromosome painting probe 18 was performed to analyze the patient and his parents. In addition, single nucleotide polymorphism-based arrays (SNP-Array) analysis with Affymetrix GeneChip Genome-wide Human SNP Nsp/Sty 6.0 were also performed to analyze the patient. RESULTS: Karyotype analysis indicated that the patient has carried a terminal deletion in 5p. FISH with Cri du Chat syndrome region probe confirmed that D5S23 and D5S721 loci are deleted. SNP-Array has detected a 15 Mb deletion at 5p and a 2 Mb duplication at 18p. FISH with 5p subtelomeric probes and 18p subtelomeric probe further confirmed that the derivative chromosome 5 has derived from a translocation between 5p and 18p, which has given rise to a 46,XY,der(5)t(5;18)(p15.1;p11.31)dn karyotype. CONCLUSION: A de novo 5p partial deletion in conjunction with a cryptic 18p duplication has been detected in a boy featuring Cri-du-Chat syndrome. His parents, both with negative findings, have a low recurrence risk. For its ability to detect chromosomal imbalance, SNP-Array has a great value for counseling of similar patients and assessment of recurrence risks.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5 , Síndrome de Cri-du-Chat/diagnóstico , Síndrome de Cri-du-Chat/genética , Trissomia , Pré-Escolar , Bandeamento Cromossômico , Cromossomos Humanos Par 18 , Humanos , Hibridização in Situ Fluorescente , Masculino , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: Mutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed. METHODS: In this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree. RESULTS: Deletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents. CONCLUSION: Two novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.
Assuntos
Testes Genéticos , Mutação/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Diagnóstico Pré-Natal , Trombastenia/diagnóstico , Trombastenia/genética , Sequência de Bases , Pré-Escolar , Feminino , Ordem dos Genes , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Gravidez , Conformação ProteicaRESUMO
OBJECTIVE: To identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. METHODS: PCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. RESULTS: Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. CONCLUSION: Six novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.
Assuntos
Fator VIII/genética , Testes Genéticos/métodos , Hemofilia A/genética , Mutação , Cromossomos Humanos X , Análise Mutacional de DNA/métodos , Enzimas de Restrição do DNA/genética , Feminino , Hemofilia A/diagnóstico , Heterozigoto , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVE: To screen mutations of tuberous sclerosis complex (TSC) patients to confirm a clinical diagnosis of TSC, and to perform prenatal diagnosis for families with mutations. METHODS: In this study, PCR-denaturing high-performance liquid chromatography(DHPLC), supplemented with sequencing when necessary, was used to screen TSC1 and TSC2 mutations in 21 patients from 19 pedigrees visited author's hospital in the last five years. For novel mutations, one hundred unrelated healthy individuals were screened to exclude the possibility of polymorphism. RESULTS: Seventeen different mutations were found in 21 patients of 19 pedigrees with 13 being novel mutations, including c. 2672delA, c. 2672insA of TSC1 gene and c.4918insCGCC, c.1143delG, Intron27+1 G>A, c.1957-1958delAG, Intron5+1 G>A, c.910insCT, c.2753 C>G, c.4078dupAGCAAGTCCAGCTCCTC, Intron 11 -1 G>A, Intron 14+1 G>A, c.684 C>A of TSC2 gene, indicating a high frequency of de novo mutations in TSC. Three of these mutations were in the TSC1 gene (N762S, c.2672insA and c. 2672delA), while all remaining 14 were in the TSC2 gene. Prenatal diagnosis for TSC was performed for 7 fetuses from these pedigrees. The six fetuses that tested negative for TSC mutations were carried to term and, to date, none of these children has shown symptoms of TSC. CONCLUSION: Author's data showed that a mutation detection rate of tuberous sclerosis was 89.5%(17/19) among patients in author's hospital. The ratio of TSC2 and TSC1 mutations was about 1:1 in the familial cases, but TSC2 mutation was more common than TSC1 mutation in sporadic cases. Author's data demonstrated that birth of TSC children for those with familial history of TSC could be prevented through prenatal diagnosis.
Assuntos
Análise Mutacional de DNA/métodos , Diagnóstico Pré-Natal/métodos , Esclerose Tuberosa/diagnóstico , Esclerose Tuberosa/genética , Sequência de Bases , Feminino , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Estudos RetrospectivosRESUMO
OBJECTIVE: To determine the karyotype of a patient with Prader-Willi-like syndrome features. METHODS: Chromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region. RESULTS: No abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient. CONCLUSION: Molecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Síndrome de Prader-Willi/genética , Criança , Feminino , Humanos , CariotipagemRESUMO
Mtsarg1 (Mus musculus testis and spermatogenesis cell apoptosis-related gene 1) gene with 1103 bp in full length had been cloned previously (GenBank accession number: AF399971, 2002; re-designated as Spata3, Mus musculus spermatogenesis-associated 3, 2007). In the present study, we identified a novel transcript variant of Mtsarg1, named Mtsarg1-beta which is 887 bp in length (GenBank accession numbers: EU259321 and EF546784, 2007, designated as Spata3 variant 4) by reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing. Mtsarg1-beta which has high similarity with Mtsarg1 contains an entire open reading frame of 417 bp encoding a protein consisting of 138 amino acids. Mtsarg1-beta protein is a non-secretory protein with a theoretic molecular mass around 14.79 kD and an isoelectric point of 9.74, which shares the 100 N-terminal amino acids with Mtsarg1 followed by 38 amino acids differing from Mtsarg1. Multi-tissue RT-PCR results and Northern blot analysis for adult DBA/2 mice showed that Mtsarg1-beta and Mtsarg1 mRNAs were specifically expressed in testis at high level. RT-PCR results also showed that Mtsarg1-beta and Mtsarg1 mRNAs were not expressed in mouse GC-1 spermatogonia. In situ hybridization revealed that both Mtsarg1 and probably Mtsarg1-beta mRNAs were mainly expressed in mouse spermatocytes. Subcellular localization analysis suggested that Mtsarg1 protein was mainly localized in nucleus while Mtsarg1-beta protein was mainly localized in cytoplasm. All these results indicate that Mtsarg1 and Mtsarg1-beta may play an important role in mouse testicular function and in spermatocyte development.
Assuntos
Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Éxons/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hibridização In Situ , Íntrons/genética , Masculino , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas/química , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogônias/metabolismo , Frações Subcelulares/metabolismoRESUMO
OBJECTIVE: To identify the mutations of the tyrosinase gene (TYR) and P gene in patients with oculocutaneous albinism (OCA). METHODS: Polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC) were applied to detect the mutations in all exons of TYR gene and P gene. Then DNA sequencing and restriction endonuclease analysis were used to confirm the mutations detected by DHPLC. Novel mutations were screened in 100 unrelated persons with normal phenotypes to exclude the possibility of polymorphism. RESULTS: Two mutations were detected in the P gene of the three patients and none in TYR gene. Heterozygous mutation of T450M in exon 13 of the P gene was detected in patient 1. Patient 2 had a heterozygous mutation of T450M in exon 13 and a heterozygous mutation of G775R in exon 23 of the P gene. Patient 3 had a heterozygous mutation of G775R as well. Restriction endonuclease analysis of the P gene exon 13 showed that the Oli I site had partly disappeared resulting from the heterozygous mutation T450M in patient 1 and patient 2, but not in 100 unrelated individuals. The heterozygous mutation T450M is a novel mutation. CONCLUSION: Gene diagnosis of OCA can be carried out effectively by combining PCR, DHPLC, DNA sequencing and restriction endonuclease analysis.
Assuntos
Albinismo Oculocutâneo/genética , Catecol Oxidase/genética , Monofenol Mono-Oxigenase/genética , Mutação , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , Éxons/genética , Feminino , Síndrome de Hermanski-Pudlak/genética , Humanos , Adulto JovemAssuntos
Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 15/genética , Infertilidade Masculina/genética , Diagnóstico Pré-Implantação/métodos , Translocação Genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Recém-Nascido , Cariotipagem , Masculino , Gravidez , Resultado da Gravidez , Medição de Risco , Análise do SêmenRESUMO
OBJECTIVE: To characterize a supernumerary marker chromosome (SMC) by comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH) and traditional cytogenetic techniques, and to explore the clinical application of these techniques in delineating de novo marker chromosomes. METHODS: A mental retardation patient received chromosome test by ordinary G banding. CGH and FISH techniques were used to analyze the origin of the de novo SMC, and N banding technique and C banding techniques were used to analyze the SMC structure. The phenotypic effects of the SMC were analyzed after the karyotype was determined. RESULTS: By G banding technique, the patient was showed to have a mosaic karyotype with SMC: mos.47, XX, +mar [31]/48, XX, +2mar[29]. CGH analysis showed a gain of 15q11 --> q14, and the result was confirmed by FISH with chromosome 15 painting probe. The further FISH analysis showed the SMC had two signals with UBE3A probe for detecting Prader-willi syndrome/Angelman syndrome (PWS/AS). N banding and C banding analysis showed the SMC had a double satellite and double centromere, respectively. Combined with the above results, the karyotype of the patient was: mos.47, XX, +der (15) (pter --> q14::q14 --> pter) [31]/48, XX, +2der (15) (pter --> q14::q14 --> pter) [29]. ish der(15)(WCP15+, UBE3A++, PML-). CONCLUSION: CGH is a valuable method to detect imbalanced chromosomal rearrangement. Combined with FISH and the traditional cytogenetic technique, it provides a valuable technique platform for characterizing the structure of the de novo SMC, and a basis for exploring the relation between karyotype and phenotype, prognosis and recurrent risk.
Assuntos
Aberrações Cromossômicas , Análise Citogenética/métodos , Citogenética/métodos , Bandeamento Cromossômico , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , CariotipagemRESUMO
OBJECTIVE: To detect gene mutation in the patients with autosomal dominant polycystic kidney disease (PKD). METHODS: Polymerase chain reaction (PCR)-denaturing high-performance liquid chromatography (DHPLC) analyses were performed in 3o single copy region of PKD 1 gene (PKD1). DNA sequencing were carried out on PCR products with abnormal peak shape afterwards. RESULTS: A new nonsense mutation (C11901A in exon 42 of PKD1 was identified to cause serine in position 3897 turning to a stop codon. A missense mutation, C10737T, was detected in exon 35 which caused threonine in position 3509 turn to methionine. Two kinds of samesense mutation, G11824A and C11860T in exon 42, were found in normal control. CONCLUSION: PKD1 mutation were detected successfully by PCR-DHPLC. A new nonsense mutation, a missense mutation and two polymorphisms are identified in this study.
Assuntos
Códon sem Sentido , Mutação de Sentido Incorreto , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Adulto , Feminino , Humanos , Masculino , Doenças Renais Policísticas/genética , Adulto JovemRESUMO
OBJECTIVE: To analyze the karyotypes of 11 cases of Turner syndrome with marker chromosome, and study the phenotypic effects resulting from the abnormal karyotype. METHODS: Eleven Turner syndrome patients had a mosaic karyotype and carried a marker chromosome, and 6 marker chromosomes were ring chromosomes. Their karyotypes were showed as mos. 45, X/46, X, + mar or mos. 45, X/46, X, + r. Fluorescence in situ hybridization (FISH) technique with X/Y centromere probes was performed to determine the origin of the marker chromosome. Reverse chromosome painting technique was used to identify the breakpoints of two largest markers. Phenotype effects with different chromosome breakpoints were compared. RESULTS: All the 11 marker chromosomes were ring X chromosomes. The breakpoints of the r(X) were involved in Xp22, Xq22, Xq24 and Xq26, etc. CONCLUSIONS: The marker chromosomes in Turner syndrome mainly originate from X chromosome and form ring chromosome X. Each r(X) in our patients was mosaic, indicating it was originated from mitosis error during early embryo development. To analyze the origin of the marker chromosome and the breakpoint of r(X) will provide guidance for the therapy and prognosis of the Turner syndrome patient.
Assuntos
Hibridização in Situ Fluorescente/métodos , Cromossomos em Anel , Síndrome de Turner/genética , Adolescente , Adulto , Criança , Pré-Escolar , Aberrações Cromossômicas , Bandeamento Cromossômico , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Feminino , Humanos , Lactente , Cariotipagem , Fenótipo , Síndrome de Turner/patologiaRESUMO
To understand the function of SRG4, a novel spermatogenesis gene, we studied its expression pattern during normal mouse development and in experimental cryptorchidism. Testis tissues were collected from 1-, 3-, and 12-week-old normal mice and immunohistochemistry was used to detect the expression of SRG4. We performed surgery on mice to cre- ate unilateral cryptorchidism and monitored SRG4 mRNA levels by semi-quantitative RT-PCR in the cryptorchid testis from day 0-18. At post-operative day 18, the cryptorchid testis and the contralateral control testis were harvested and assayed for SRG4 expression by in situ hybridization. Immunohistochemistry results showed that SRG4 protein was hardly detected in 1-week-old mouse testis, but the expression was present in 3-week-old mouse and abundant in 12-week-old mouse testis. SRG4 immunostaining was mainly localized to the cytoplasm and membrane of spermatocytes and round spermatids. Moreover, semi-quantitative RT-PCR result showed the expression of SRG4 mRNA did not decrease until 9 d after cryptorchid surgery, and continued to decline thereafter. In situ hybridization revealed that in contrast to the abundant SRG4 expression in the control side, few remaining germ cells in the crytorchid testis were positive for SRG4 at d 18 after surgery. The results indicated that the expression of SRG4 was regulated by development, and SRG4 was mainly expressed in the cytoplasm of spermatocytes and round spermatids. However, in cryptorchid testis, in which most germ cells undergo apoptosis, only a few of SRG4 is observed, suggesting that SRG4 may be as a specific marker to evaluate the process of spermatogenesis.
Assuntos
Criptorquidismo/genética , Proteínas/genética , Espermatogênese/genética , Testículo/metabolismo , Animais , Criptorquidismo/metabolismo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the mTSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2,463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56,295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.
Assuntos
DNA Complementar/genética , Glicerol-3-Fosfato O-Aciltransferase/genética , Espermatogênese/genética , Testículo/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Humanos Par 8 , Clonagem Molecular , Citoplasma/metabolismo , Feto , Glicerol-3-Fosfato O-Aciltransferase/biossíntese , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Maturidade Sexual/genética , Temperatura , Testículo/crescimento & desenvolvimentoRESUMO
OBJECTIVE: To identify the mutation in transforming growth factor-beta1 gene (TGF beta1) in a Chinese patient with Camurati-Engelmann disease(CED). METHODS: Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on the whole seven coding exons and exon-intron boundaries, then the mutation was identified by direct sequencing. RESULTS: Mutation screening of TGF beta1 in this patient revealed a heterozygous missense mutation R218H in exon 4. CONCLUSION: The identification of the mutation could provide essential data for subsequent therapy and genetic counseling.
Assuntos
Síndrome de Camurati-Engelmann/genética , Mutação , Fator de Crescimento Transformador beta1/genética , Sequência de Bases , China , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Humanos , Masculino , Reação em Cadeia da Polimerase , Adulto JovemRESUMO
OBJECTIVE: To analyze the mutations in Cx30 gene in a Chinese family with hidrotic ectodermal dysplasia (HED) and to make prenatal diagnosis on the embryo which has been pregnant for 5 months. METHODS: A family including 2 affected and 4 unaffected individuals was collected, and their informed consents were obtained. The affected woman had a five-month pregnancy. An 884 bp fragment containing the whole GJB6 coding sequence was amplified by PCR and the products were bi-direction sequenced directly. The mutation was further confirmed with restriction endoenzyme digesting. On the base of successful gene diagnosis, the following detection procedure on the pregnant baby was performed. First the whole coding region of Cx30 was amplified using primers Cx30-F and Cx30-R and the PCR products were digested by Hae II. Then the PCR products were cloned into pUCm-T vector. Blue-white blot screening method and PCR-restriction endoenzyme digesting technique were used to identify the correct clones. The mutant allele clone was sequenced to confirmed the mutation. RESULTS: A heterozygous missense mutation 263C --> T in the Cx30 gene was detected in the affected little girl and her affected mother, which led to an amino acid substitution (A88V) in the second transmembrane domain of GJB6. The mutation was confirmed by Hae II digestion. A88V mutant allele cannot be cut while the wild normal allele can be cut into two fragments, 520 and 278 bp. The result of analyse on the five-month pregnancy show the embryo carried the A88V mutation too. So the embryo will be a patient. CONCLUSION: An A88V missense mutation in the Cx30 gene can also cause HED in Chinese Han population. Based on the gene diagnosis, prenatal diagnosis can be played using bi-direction sequencing and confirmed with restriction endoenzyme digesting.
Assuntos
Conexinas/genética , Displasia Ectodérmica/genética , Doenças Fetais/genética , Mutação de Sentido Incorreto , Diagnóstico Pré-Natal/métodos , Adulto , Substituição de Aminoácidos , Povo Asiático/genética , China , Conexina 30 , Displasia Ectodérmica/etnologia , Feminino , Doenças Fetais/diagnóstico , Doenças Fetais/etnologia , Testes Genéticos , Heterozigoto , Humanos , Linhagem , Gravidez , Análise de Sequência de DNARESUMO
In order to assess the value of applying human chromosomal telomeric band painting probes (TBPs) in genetic diagnosis, we used three human telomeric band painting probes in fluorescence in situ hybridization (FISH) to analyze two cases who had a history of habitual abortion and were suspected to carrying a minute translocation. The probes were prepared by microdissection, and the covered regions were 11q23.3-->qter12q24.1-->qter and 22q13.1-->qter, respectively. According to the FISH results, both the cases were cryptic minute translocation carriers. The involved chromosomes were chromosome 11 and 12 in case 1, and chromosome 11 and 22 in case 2. Combined with chromosome G banding, their breakpoints were 11q23.3, 12q24.1, and 22q13.1, respectively. The results showed that the telomeric band painting probes can be used as a tool to determine minute chromosomal abnormality and the precise breakpoints.
Assuntos
Aborto Habitual/genética , Telômero/genética , Translocação Genética , Adulto , Bandeamento Cromossômico , Coloração Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 2/genética , Cromossomos Humanos Par 22/genética , Feminino , Humanos , MasculinoRESUMO
A novel mouse zinc finger protein gene that contains C2HC/C3H domain was first isolated by using a data-mining tool called Digital Differential Display (DDD) from the National Center for Biotechnology Information. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Total four exons of the mouse gene spaning a 29 869 bp genomic DNA sequence was mapped to chromosome 18D1. The cDNA encodes a novel protein of 347 amino acids and the protein contains four C2HC/C3H domains. Northern blot analyses revealed that Zfp474 mRNA was exclusively expressed in testis and ovary and had one transcript. We hypothesize that Zfp474 functions as a germ cell specific transcription factor that plays important roles in spermatid differentiation and oocyte development.
Assuntos
DNA Complementar/genética , Ovário/metabolismo , Testículo/metabolismo , Fatores de Transcrição/biossíntese , Dedos de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Etiquetas de Sequências Expressas , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espermatogônias/citologia , Espermatogônias/metabolismo , Distribuição Tecidual , Fatores de Transcrição/genéticaRESUMO
The SRY gene (sex determining region on Y chromosome) acts as TDF and is required for regulating male sex determination. SRY represents a transcription factor belonging to the superfamily of genes sharing the HMG-box motif (high-mobility group-box), which acts as DNA binding region. Deletion and inactivating mutations of SRY are among the known causes of XY sex reversal. Here, we described the screening of 10 patients who presented with 46,XY sex reversal for mutations in open reading frame (ORF) of SRY gene. DNA was isolated from blood samples using standard techniques. A 609 bp fragment from the central portion of the SRY gene was amplified, using primers XES-2 and XES-7. The amplified PCR fragments were cloned into the pUCm-T vectors, and direct sequencing were carried out on an ABI 377-3 automated DNA sequencer to detect the mutation. PCR-restriction enzyme digestion was applied to detect the results of DNA sequencing. In two patients,de novo mutations led to an amino acid substitution. An A was replaced by a G in codon 38 upstream of the 5' border outside the HMG box of the SRY gene, resulting in the replacement of the amino acid glutamate by glycine. Another heterozygous T to A transition at the nucleotide position +387 which encodes for a Tyrosine (Tyr) instead of a Term, whereas her father was proven to have the wild-type sequence. These point mutations have been confirmed with PCR-restrict enzyme method. As demonstrated by the Human Gene Mutation Database analysis,homology search, and review of the literature, these two mutations were not described previously and brought the total number of SRY gene nucleotide substitutions (missense/nonsense) to 45. These findings indicated that these amino acid substitutions may be responsible for the sex reversal,not only inside the HMG-box but also outside the HMG-box. The two novel mutations in SRY gene provided valuable information for understanding the molecular mechanism of the patient with 46,XY female sex reversal.
Assuntos
Transtornos do Desenvolvimento Sexual , Genes sry , Disgenesia Gonadal 46 XY/genética , Mutação Puntual , Substituição de Aminoácidos , Povo Asiático/genética , Sequência de Bases , China , DNA/química , DNA/genética , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Feminino , Disgenesia Gonadal 46 XY/etnologia , Humanos , Masculino , Fases de Leitura Aberta , Análise de Sequência de DNARESUMO
OBJECTIVE: To explore the influence factors on amplification of single cell duplex-nested PCR. METHODS: The mutational loci region CD41-42 and IVS-II 654 of beta-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 degrees C before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR. RESULTS: TaKaRa EX Taq was the most efficient Taq DNA polymerase among different Taq DNA polymerases; primer pair R1+F1 at final concentration of 0.25 micromol/L and R2+F2 at 0.3 micromol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P<0.05); there were no remarkable differences of the amplification efficiency while using whether predenaturation at 98 degrees C before the single cell PCR amplification or not (P>0.05). CONCLUSION: There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 degrees C before the single cell PCR amplification could not improve the PCR amplification efficiency.