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BACKGROUND: Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis. METHODS: Eighty-one HTX patients with complex congenital heart defects and 89 healthy children were enrolled, and we investigated the pathogenetic variants related to patients with HTX by exome sequencing. Zebrafish splice-blocking Morpholino oligo-mediated transient suppression assays were performed to confirm the potential pathogenicity of missense variants found in these patients with HTX. RESULTS: Three MMP21 heterozygous non-synonymous variants (c.731G > A (p.G244E), c.829C > T (p.L277F), and c.1459A > G (p.K487E)) were identified in three unrelated Chinese Han patients with HTX and complex congenital heart defects. Sanger sequencing confirmed that all variants were de novo. Cell transfection assay showed that none of the variants affect mRNA and protein expression levels of MMP21. Knockdown expression of mmp21 by splice-blocking Morpholino oligo in zebrafish embryos revealed a heart looping disorder, and mutant human MMP21 mRNA (c.731G > A, c.1459A > G, heterozygous mRNA (wild-type&c.731G > A), as well as heterozygous mRNA (wild-type& c.1459A > G) could not effectively rescue the heart looping defects. A patient with the MMP21 p.G244E variant was identified with other potential HTX-causing missense mutations, whereas the patient with the MMP21 p.K487E variant had no genetic mutations in other causative genes related to HTX. CONCLUSION: Our study highlights the role of the disruptive heterozygous MMP21 variant (p.K487E) in the etiology of HTX with complex cardiac malformations and expands the current mutation spectrum of MMP21 in HTX.
Assuntos
Síndrome de Heterotaxia , Animais , Criança , China , Síndrome de Heterotaxia/genética , Humanos , Morfolinos , RNA Mensageiro , Fatores de Risco , Peixe-Zebra/genéticaRESUMO
Purpose: This study aimed at exploring the feasibility and reproducibility of CCT for the measurement of Left Atrial (LA) strain and volume compared with transthoracic echocardiography (TTE) in pediatric patients with congenital heart disease (CHD). Materials and Methods: The present study included 43 postoperative patients with CHD (7.39 ± 3.64 years, 56% male) who underwent clinically indicated CCT, and all patients underwent additional TTE on the same day. LA strain and volume parameters were measured by dedicated software. The correlation and agreement of LA strain and volume parameters were assessed using Pearson's correlation coefficient and Bland-Altman analysis. Intra-class correlation coefficients (ICC) were used to assess CCT intra-observer and inter-observer reproducibility. Results: All strain parameters of CCT were lower compared to TTE (reservoir strain: 28.37 ± 6.92 vs. 32.15 ± 8.15, respectively; conduit strain: 21.33 ± 6.46 vs. 24.23 ± 7.75, respectively; booster strain: 7.04 ± 2.74 vs. 7.92 ± 3.56). While the volume parameters of CCT were higher compared to TTE (LAV: 29.60 ± 19.01 vs. 25.66 ± 17.60, respectively; LAVi: 30.36 ± 22.31 vs. 28.63 ± 19.25, respectively). Both LA strain and volume measurements showed good correlation and agreement between the two modalities (r = 0.63-0.87, p < 0.001). CT-derived LA strain and volume measurements showed good intra- and inter-observer reproducibility using prototype software (ICC = 0.78-0.96). Conclusions: CCT was feasible for measuring LA strain and volume with good correlation and high reproducibility as compared with TTE. As a complementary modality, CCT can regard as an accepted method in the evaluation of LA function in pediatric patients with CHD.
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BACKGROUND: SmD1-amino-acid 83-119 peptide (SmD183-119) is the major epitope of Smith (Sm) antigen, which is specific for adult systemic lupus erythematosus (SLE). The anti-SmD183-119 antibody has exhibited higher sensitivity and specificity than anti-Sm antibody in diagnosing adult SLE. However, the utility of anti-SmD183-119antibodies remains unclear in children with SLE (cSLE). This study aimed to assess the characteristics of anti-SmD183-119antibody in the diagnosis of cSLE. METHODS: Samples from 242 children with different rheumatological and immunological disorders, including autoimmune diseases (SLE [n = 46] and ankylosing spondylitis [AS, n = 11]), nonautoimmune diseases (Henoch-Schonlein purpura [HSP, n = 60], idiopathic thrombocytopenia purpura [n = 27], hematuria [n = 59], and arthralgia [n = 39]) were collected from Shanghai Children's Medical Center from March 6, 2012 to February 27, 2014. Seventy age- and sex-matched patients were enrolled in this study as the negative controls. All the patients' sera were analyzed for the anti-SmD183-119, anti-Sm, anti-U1-nRNP, anti-double-stranded DNA (dsDNA), anti-nucleosome, anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB, anti-Scl-70, and anti-histone antibodies using the immunoblotting assay. The differences in sensitivity and specificity between anti-SmD183-119 and anti-Sm antibodies were compared by Chi-square test. The correlations between anti-SmD183-119and other auto-antibodies were analyzed using the Spearman's correlation analysis. A value of P< 0.05 was considered statistically significant. RESULTS: Thirty-six out of 46 patients with cSLE were found to be positive for anti-SmD183-119, while 12 patients from the cSLE cohort were found to be positive for anti-Sm. Compared to cSLE, it has been shown that anti-SmD183-119 was only detected in 27.3% of patients with AS and 16.7% of patients with HSP. In comparison with anti-Sm, it has been demonstrated that anti-SmD183-119 had a higher sensitivity (78.3% vs. 26.1%, χ2 = 25.1, P< 0.05) and a lower specificity (90.8% vs. 100%, χ2 = 13.6, P< 0.05) in the diagnosis of cSLE. Further analysis revealed that anti-SmD183-119antibodies were positively correlated with anti-dsDNA, anti-nucleosome, and anti-histone antibodies in cSLE. Moreover, it has been clearly shown that anti-SmD183-119 was more sensitive than anti-Sm in discriminating autoimmune diseases from nonautoimmune disorders in patients with arthralgia or hematuria. CONCLUSIONS: Measurement of anti-SmD183-119in patients with cSLE has a higher sensitivity and a marginally lower specificity than anti-Sm. It has been suggested that inclusion of anti-SmD183-119testing in the integrated laboratory diagnosis of cSLE may significantly improve the overall sensitivity in child populations.
Assuntos
Doenças do Sistema Imunitário/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Peptídeos/imunologia , Proteínas Centrais de snRNP/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Criança , Feminino , Humanos , Immunoblotting , Masculino , Peptídeos/químicaRESUMO
BACKGROUND: TBX1 and CRKL haploinsufficiency is thought to cause the cardiac phenotype of the 22q11.2 deletion syndrome. However, few unequivocal mutations of TBX1 and CRKL have been discovered in isolated conotrucal heart defects (CTDs) patients. The aim of the study was to screen the mutation of TBX1 and CRKL in isolated CTDs Chinese patients without 22q11.2 deletion and identify the pathomechanism of the missense mutations. METHODS: We enrolled 199 non-22q11.2 deletion patients with CTDs and 139 unrelated healthy controls. Gene sequencing were performed for all of them. The functional data of mutations were obtained by in vitro transfection and luciferase experiments and computer modelling. RESULTS: Screening of the TBX1 coding sequence identified a de novo missense mutation (c.385G â A; p.E129K) and a known polymorphism (c.928G â A; p.G310S). In vitro experiments demonstrate that the TBX1E129K variant almost lost transactivation activity. The TBX1G310S variant seems to affect the interaction of TBX1 with other factors. Computer molecular dynamics simulations showed the de novo missense mutation is likely to affect TBX1-DNA interaction. No mutation of CRKL gene was found. CONCLUSIONS: These observations suggest that the TBX1 loss-of-function mutation may be involved in the pathogenesis of isolated CTDs. This is the first human missense mutation showing that TBX1 is a candidate causing isolated CTDs in Chinese patients without 22q11.2 deletion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Síndrome de DiGeorge/genética , Cardiopatias Congênitas/genética , Proteínas Nucleares/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Povo Asiático/genética , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , DNA/química , DNA/metabolismo , Síndrome de DiGeorge/patologia , Éxons , Feminino , Células HEK293 , Cardiopatias Congênitas/patologia , Humanos , Masculino , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Filogenia , Conformação Proteica , Estrutura Secundária de Proteína , Análise de Sequência de DNA , Proteínas com Domínio T/químicaRESUMO
Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G>A (p.W13X) nonsense mutation and c.597C>G (p.I199 M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199 M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Isovaleril-CoA Desidrogenase/genética , Mutação de Sentido Incorreto , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Sequência de Aminoácidos , Povo Asiático/genética , Estabilidade Enzimática , Feminino , Flavinas/metabolismo , Heterozigoto , Humanos , Recém-Nascido , Isovaleril-CoA Desidrogenase/deficiência , Isovaleril-CoA Desidrogenase/metabolismo , Dados de Sequência Molecular , Mapeamento de Interação de ProteínasRESUMO
BACKGROUND: Wilson disease (WND), also called hepatolenticular degeneration, is an autosomal recessive genetic disorder in which copper abnormally accumulates in several organs. WND arises from the defective ATP7B gene, which encodes a copper transporting P-type ATPase. METHODS: The molecular defects in 11 unrelated Chinese WND patients aged from 3 to 12 years were investigated. The diagnosis of these patients was based on typical clinical symptoms and laboratory testing results. All 21 exons and exon-intron boundaries of the ATP7B gene were amplified by polymerase chain reaction from the genomic DNA of the patients and then analyzed by direct sequencing. One hundred healthy subjects served as controls to exclude gene polymorphism. RESULTS: In one novel (c.3605 C>G) and nine recurrent mutations of ATP7B identified, there were eight missense mutations, one splice-site mutation, and one nonsense mutation. The novel c.3605 C>G mutation resulted in the substitution of alanine by glycine at amino acid position 1202 (p.Ala1202Gly). The most frequent ATP7B mutation was c.2333 G>T (p.Arg778Leu), followed by c.2975 C>T (p.Pro992Leu), which accounted for 63.6% of the WND mutated alleles. CONCLUSIONS: The novel c.3605 C>G mutation in. ATP7B is one of the molecular mechanisms of WND.
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Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Degeneração Hepatolenticular/genética , Mutação , Criança , Pré-Escolar , ATPases Transportadoras de Cobre , HumanosRESUMO
OBJECTIVE: To investigate the role of the MAMLD1 gene mutation in the pathogenesis of hypospadias in the Chinese population. METHODS: We collected peripheral venous blood from 150 Chinese children with hypospadias (the case group) and another 120 normal healthy ones (the control group), aged 0.5 to 6 years. We obtained their DNA samples and performed DNA sequencing on the single-nucleotide polymorphisms of MAMLD1, followed by comparative analysis. RESULTS: A known missense mutation polymorphism p. N589S was identified in 12 (8.0%) of the hypospadias patients and 4 (3.0%) of the normal controls, and a novel missense mutation polymorphism p. N567S was identified in 4 (2.7%) of the patients and 3 (2.5%) of the controls, neither with statistically significant differences between the two groups (P > 0.05). CONCLUSION: The results re-emphasized the importance of replication in genetic association approaches, and might reveal a real difference in susceptibility genes among different populations. The single-nucleotide polymorphisms of MAMLD1 bear no obvious correlation with hypospadias, and MAMLD1 is not a candidate gene in its pathogenesis in the Chinese population.
Assuntos
Proteínas de Ligação a DNA/genética , Hipospadia/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Povo Asiático/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Frequência do Gene , Haplótipos , Humanos , Lactente , MasculinoRESUMO
OBJECTIVE: To investigate the incidence of inherited protein C deficiency in the patients with venous thromboembolism (VTE). METHODS: From Apr. of 2010 to Apr. of 2011, 106 patients with VTE totally from Renji hospital were surveyed by a series of laboratory tests including clinical biochemistry tests, coagulation factors activities and anticoagulation factors activities. PROC gene mutations were screened by PCR-direct sequencing in the 20 patients with decreased PC activity. RESULTS: Among the 20 patients with decreased PC activity, the median activity of factor II, V, VII, VIII, IX, X, XI, XII were 97.0%, 199.9%, 105.5%, 254.7%, 106.4%, 150.4%, 123.1%, 89.9%, respectively.6 PROC gene mutations were found in 11 patients. Six patients have the same point mutation (c.565C > T), the other five mutations were c.508G > T, c.524G > A, c.1174G > A, c.1157T > C, c.577-579del. All of the six mutations were heterozygous, while the c.508G > T, c.524G > A and c.1157T > C were novel in the world. CONCLUSIONS: In this study, we found that PC deficiency is the major inherited risk factor of VTE. The most common PROC mutation identified in this study was heterozygous c.565C > T missense mutation., c.508G > T, c.524G > A and c.1157T > C were novel PROC mutation. The activities of factor V and VIII were elevated dramatically among VTE patients, which may be correlated to the disease.
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Deficiência de Proteína C/genética , Tromboembolia Venosa/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores de Coagulação Sanguínea/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Proteína C/genética , Deficiência de Proteína C/complicações , Tromboembolia Venosa/etiologia , Adulto JovemRESUMO
BACKGROUND: Conotruncal heart defects (CTDs) are present in 75-85% of patients suffering from the 22q11.2 deletion syndrome. To date, no consistent phenotype has been consistently correlated with the 22q11.2 deletions. Genetic studies have implicated TBX1 as a critical gene in the pathogenesis of the syndrome. The aim of study was to determine the incidence of the 22q11.2 deletion in Chinese patients with CTDs and the possible mechanism for pathogenesis of CTDs. METHODS: We enrolled 212 patients with CTDs and 139 unrelated healthy controls. Both karyotypic analysis and multiplex ligation-dependent probe amplification were performed for all CTDs patients. Fluorescence in situ hybridization was performed for the patients with genetic deletions and their relatives. The TBX1 gene was sequenced for all patients and healthy controls. The χ2 and Fisher's exact test were used in the statistical analysis. RESULTS: Thirteen of the 212 patients with CTDs (6.13%) were found to have the 22q11.2 deletion syndrome. Of the 13 cases, 11 presented with a hemizygous interstitial microdeletion from CLTCL1 to LZTR1; one presented with a regional deletion from CLTCL1 to DRCR8; and one presented with a regional deletion from CDC45L to LZTR1. There were eight sequence variants in the haploid TBX1 genes of the del22q11 CTDs patients. The frequency of one single nucleotide polymorphism (SNP) in the del22q11 patients was different from that of the non-del patients (P < 0.05), and the frequencies of two other SNPs were different between the non-del CTDs patients and controls (P < 0.05). CONCLUSIONS: CTDs, especially pulmonary atresia with ventricular septal defect and tetralogy of Fallot, are the most common disorders associated with the 22q11.2 deletion syndrome. Those patients with both CTDs and 22q11.2 deletion generally have a typical or atypical deletion region within the TBX1 gene. Our results indicate that TBX1 genetic variants may be associated with CTDs.
Assuntos
Povo Asiático/genética , Síndrome de DiGeorge/diagnóstico , Síndrome de DiGeorge/genética , Cardiopatias Congênitas/genética , Polimorfismo de Nucleotídeo Único , Proteínas com Domínio T/genética , Povo Asiático/estatística & dados numéricos , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Análise Citogenética , Síndrome de DiGeorge/complicações , Síndrome de DiGeorge/epidemiologia , Feminino , Estudos de Associação Genética , Loci Gênicos , Haploidia , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/epidemiologia , Cardiopatias Congênitas/etnologia , Humanos , Incidência , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único/fisiologia , Proteínas com Domínio T/análiseRESUMO
OBJECTIVE: To identify novel genetic mutations in Chinese patients with congenital patent ductus arteriosus (PDA). METHOD: Clinical data and peripheral blood specimens from a kindred spanning 3 generations in which 5 of 16 individuals had PDA and a cohort of 95 unrelated subjects with PDA were collected, and 100 unrelated healthy individuals were included as controls. The coding exons and flanking introns of TFAP-2B gene were amplified by polymerase chain reaction (PCR) with specific primers. We aligned the acquired sequences with which publicized in GenBank by the aid of program BLAST. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the parts of TFAP-2B and sequencing was performed on PCR products forward and reversely directly. RESULT: Sequencing of TFAP-2B identified that there was a splice-junction in intron 3 [intron 3(+5)G > A] and a 60 bp deletion was found in exon 3 by nested PCR. Additionally, a novel single nucleotide polymorphism (SNP) where a transition of guanine (G) to adenine (A) was identified at 34 bp front of transcription initiation site in TFAP-2B gene. There were significant differences in the prevalence of alleles G and A between controls and PDA patients (Z = -2.513, P = 0.012). CONCLUSION: We identified a novel splice-junction in TFAP-2B gene which might lead to hereditary PDA in a Chinese family. However, the mechanism by which this mutation results in PDA is still to be ascertained.
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Permeabilidade do Canal Arterial/genética , Mutação , Fator de Transcrição AP-2/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Éxons , Feminino , Humanos , Lactente , MasculinoRESUMO
AIMS: To investigate the molecular defects in a Chinese pedigree with inherited factor V (FV) deficiency. METHODS: Laboratory studies including activated partial thromboplastin time (APTT), prothrombin (PT), and thrombin time (TT) were tested in a patient and his family members. FV antigen (FV:Ag) and FV activity (FV:C) were measured by both ELISA and one-stage clotting assays. All the exons, exon-intron boundaries and promoter regions of FV gene were analysed by direct sequencing. The detected mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA and the wild-type and mutant FV proteins were expressed in COS-7 and CHO cells. RESULTS: The proposita, a 52-year-old Chinese man, had no spontaneous bleeding syndrome. It was found that he had prolonged APTT and PT, 52 s and 22.8 s, respectively, a FV:C of 5.5% and a FV:Ag of 33.1%. Gene analysis showed the proposita was a compound heterozygote of FV mutations, carrying Ser234Leu and Arg413Cys. The FV antigen and activity levels of the Ser234Leu and Arg413Cys mutants are lower than wild type both in cell lysates and in culture media. Protein degradation inhibitor experiment in transfected COS-7 cells showed that Ser234Leu and Arg413Cys degraded intracellularly through the lysosomal pathway. CHO cells expressing either the wild-type or the mutant FV were subjected to immunofluorescence staining with the indicated antibodies and organelle markers, indicating that Ser234Leu and Arg413Cys can be transported to Golgi partially. CONCLUSIONS: We identified the molecular pathological mechanism of the novel C785T mutation causing type I inherited FV deficiency for the first time.
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Deficiência do Fator V/genética , Fator V/genética , Leucina/genética , Mutação de Sentido Incorreto , Serina/genética , Substituição de Aminoácidos , Animais , Povo Asiático/genética , Testes de Coagulação Sanguínea , Western Blotting , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cricetinae , Cricetulus , Análise Mutacional de DNA , Fator V/análise , Deficiência do Fator V/sangue , Deficiência do Fator V/fisiopatologia , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , LinhagemAssuntos
Coagulação Sanguínea/genética , Deficiência do Fator V/genética , Fator V/genética , Animais , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Éxons , Fator V/química , Fator V/metabolismo , Deficiência do Fator V/sangue , Feminino , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Pessoa de Meia-Idade , Peso Molecular , Linhagem , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Índice de Gravidade de Doença , TransfecçãoRESUMO
OBJECTIVE: To study the molecular mechanisms of antithrombin (AT) deficiency caused by AT gene mutations T98I and A404T. METHODS: Wild-type and mutant AT cDNA expression plasmids (ATwt, AT T98I and AT A404T) were constructed and transfected into the monkey fibroblast of the line COS-7 or Chinese hamster ovary (CHO) cells. NH4Cl. ALLN and brefeldin A were added. ELISA was used to detect the AT: Ag. Pulse-chase experiment and immunofluorescence assay were used to detect the radioactivity of the 35S-labeled AT. Fluorescence real-time PCR was used to detect the expression of AT mRNA, protein degradation inhibition was used to elucidate the mutant T98I degradation pathway inside the cells. RESULTS: AT T98I was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of AT A404T, but most of the molecule was not secreted but was degraded intracellularly. Fluorescence real-time PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Protein degradation inhibition experiment showed that mutant AT T98I was degraded intracellularly through the proteasome pathway. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT T98I mutant were stained diffusely without perinuclear enhancement and cells expressing AT A404T mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. CONCLUSION: Impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular mechanism of AT deficiency caused by T98I and A404T mutation.
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Deficiência de Antitrombina III/genética , Antitrombinas/genética , Mutação , Animais , Antitrombinas/metabolismo , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
In order to establish a genotyping method for DEL phenotype in Zhejiang Han population, an AS-PCR method was developed according to the RHD 1227A allele sequence. Its specificity and sensitivity were assessed in two Rh negative populations whose RHD 1227A or DEL phenotype status was known. The results showed that in evaluation of the method by detecting 50 RHD 1227A positive and 50 RHD 1227A negative individuals, the genotyping method displayed a sensitivity of 100% and a specificity of 100%; in evaluation of the method by detecting 33 DEL positive and 89 DEL negative individuals, the sensitivity was 100%, however, there were two serologically negative samples which were confirmed as positive using genotyping method. After re-testing these two samples with serological method and sequence analysis, it was found that original serological method gave false negative results and genotyping method still showed 100% specificity. The minimal target DNA concentration of this genotyping method is 8.13 ng/microl. In conclusion, designed genotyping method can be used to identify DEL phenotype efficiently in Zhejiang Han Rh negative population.
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Eritrócitos/imunologia , Polimorfismo Genético , Sistema do Grupo Sanguíneo Rh-Hr/genética , Adulto , Alelos , Povo Asiático/genética , Doadores de Sangue , China/etnologia , Eritrócitos/metabolismo , Feminino , Genótipo , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase/métodos , Sistema do Grupo Sanguíneo Rh-Hr/imunologiaRESUMO
This study was purposed to investigate the molecular basis of Rh DEL phenotype. Rh DEL phenotypes were identified by a serologic adsorption-elution method, the nucleotide sequences of ten RHD exons and exon-intron boundary regions were evaluated by a RHD gene-specific PCR-SSP (PCR-SSP, polymerase chain reaction-sequence specific primer) and sequencing. The results showed that out of 122 random Rh negative donors 35 Rh DEL phenotypes were identified through serologic method, including 6 RhCCdee (17.14%), 28 RhCcdee (80.00%), and 1RhCcdEe (2.86%). Sequence analysis indicated that all DEL phenotypes harbored a RHD 1227 G > A mutation in exon 9. D zygosity test revealed that 29 DEL phenotypes (28 RhCcdee and 1 RhCcdEe) had one RHD gene deleted, and 6 DEL phenotypes (6 RhCCdee) had homogenous RHD gene. It is concluded that RHD 1227A is an important genetic marker for Rh DEL phenotype in Zhejiang Han population.
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Eritrócitos/imunologia , Mutação Puntual , Polimorfismo Genético , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Povo Asiático/genética , Sequência de Bases , Doadores de Sangue , China/etnologia , Éxons/genética , Humanos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase/métodos , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Análise de Sequência de DNARESUMO
The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467C > T and 539G > C) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.
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Sistema ABO de Grupos Sanguíneos/genética , Alelos , Povo Asiático/genética , Fucosil Galactose alfa-N-Acetilgalactosaminiltransferase/genética , Mutação Puntual , Sequência de Bases , China/etnologia , Feminino , Genótipo , Humanos , Masculino , Dados de Sequência MolecularRESUMO
OBJECTIVE: To investigate the mechanism of clinical haemorrhage in an inherited coagulation factor VII (FVII) deficiency and tissue factor abnormality pedigree. METHODS: All exons, exon-intron boundaries and the 3', 5' untranslated sequences of FVII and tissue factor (TF) genes were amplified by PCR and sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. FVII cDNA of the proband was synthesized with random primers and amplified by nest PCR. RESULTS: 55C-->T heterozygous mutation located in promoter of FVII gene was identified in the proband. The heterozygous mutation was derived from his mother. Tracing the other pedigree members found that his sister had the same heterozygous mutation and the others had wild-type FVII genes. A 9363 C-->T (Arg131Trp) heterozygous polymorphism in TF gene, which was 2.63% frequency of T allele polymorphism, was found in all of the pedigree members. CONCLUSION: It was the first report that the -55C-->T heterozygous mutation in FVII gene and the Arg131Trp heterozygous polymorphism in TF gene explained the clinical symptom of the proband.
Assuntos
Deficiência do Fator VII/genética , Fator VII/genética , Tromboplastina/genética , Adulto , Análise Mutacional de DNA , Heterozigoto , Humanos , Masculino , Linhagem , Polimorfismo GenéticoRESUMO
To investigate the molecular basis of partial D phenotypes in Chinese, D variants with weak D expression was screened by using indirect anti-human globulin test (IAT) method, the polymerase chain reaction-sequence specific primer (PCR-SSP) method was employed to amplify RHD specific exons and their flanking regions. The amplification products were sequenced directly to determine the molecular basis of D variants. The results showed that ten cases of partial D phenotypes, including one case of D Va (Kou.), one case of D Va (Hus.), one case of D Va-like (YH.), and seven cases of D VI type III, were detected from 22 cases of weak D phenotype respectively. All ten cases of partial D phenotypes had one RHD allele deleted. In conclusion, the molecular basis of ten cases of partial D phenotype was confirmed, including D Va (Kou.) and D Va-like (YH.) phenotypes reported firstly in Chinese population.
Assuntos
Alelos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Povo Asiático , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação , FenótipoRESUMO
To investigate the alpha-1, 3/4-fucosyltransferase gene (FUT3) polymorphism associated with Lewis blood group in Zhejiang population, the Lewis phenotypes of 183 random samples from Chinese blood donors in Zhejiang province were identified by standard serological techniques. The entire coding region of FUT3 gene were amplified by PCR from genomic DNA of 39 Lewis negative and 9 Lewis positive phenotype samples and sequenced directly. The haplotypes of FUT3 allele were identified by TOPO cloning sequencing method. The results showed that the frequency of true Le (a-b-) phenotype in Zhejiang population was 10.4% according to serological and molecular biological methods. Five nucleotide acid variant sites (59T > G, 202T > C, 314C > T, 508G > A and 1067T > A) were detected in all 48 sequencing samples. Besides the wild type Le allele, 2 common (le(59, 1067) and le(59, 508) and 3 rare non-functional le alleles (le(59), le(1067) and le(202, 314) were found in this population. In conclusion, the polymorphism of non-functional FUT3 allele was found to be relatively variable in Chinese Zhejiang population.
Assuntos
Alelos , Fucosiltransferases/genética , Antígenos do Grupo Sanguíneo de Lewis/genética , Polimorfismo Genético , Adulto , Sequência de Bases , China/etnologia , Feminino , Humanos , Masculino , Dados de Sequência MolecularRESUMO
OBJECTIVE: To investigate the h4 allele (C35T) frequency of alpha-1,2-fucosyltransferase gene in Chinese population. METHODS: The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying C35T variant was established by using PCR to amplify a 125 bp FUT1 gene fragment, including C35T variant sequence, and the PCR product was digested by Hae III restriction enzyme. One hundred and fifty-eight random samples from Chinese blood donor were screened by PCR-RFLP. RESULTS: Among 158 Chinese individuals with normal ABO and H blood group phenotypes, 8 and 83 were homozygous with 35T/T and 35C/C, respectively, while 67 were heterozygous with 35C/T. The allele frequencies were compatible with Hardy-Weinberg equilibrium. CONCLUSION: The C35T substitution of FUT1 gene is not a mutation which gives rise to a non-functional h allele responsible for para-Bombay phenotype but a single nucleotide polymorphism in Chinese population.