OBJECTIVETo explore the clinical characteristics and genetic mutations in two children with Omenn syndromes.

METHODSPeripheral venous blood samples were collected from 2 children suspected with severe combined immunodeficiency (SCID) and their family members. The samples were subjected to RAG1 and RAG2 gene sequencing and TCR Vβ subclone analysis.

RESULTSBoth patients had recurrent infections, erythroderma rashes and alopecia baldness. One patient has fit with immunophenotype T-B-NK+, while another was consistent with typical Omenn syndrome combined with T+B-NK+ immunophenotype, IgE and eosinophil increase. Both children have carried compound heterozygous mutations of the RAG1 gene. The first patient carried c.1328 G>A (p.R443K) and c.2486-2490delGGAAA (p.R829fsX869) mutations, both were of de novel type. The second patient has carried c.1209C>T (p.R403W) and c.2892delT (p.ASN964LYSfs*14), with c.2892delT (p.ASN964LYSfs*14) being a de novel mutation. The parents of both patients were heterozygous carriers. The same mutations were not found in 100 healthy children. Both patients' 24 TCR Vβ subfamilies have presented monoclonal or oligoclonal peaks, with TCR Vβ polymorphism being severely disrupted.

CONCLUSIONThree novel mutations have been identified in two children with Omenn syndrome, which featured early onset and rapid progression. Early recognition of the disease and prompt treatment may reduce the mortality.

Adult ; Base Sequence ; DNA-Binding Proteins ; genetics ; Female ; Heterozygote ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Male ; Molecular Sequence Data ; Mutation ; Nuclear Proteins ; genetics ; Pedigree ; Severe Combined Immunodeficiency ; genetics

OBJECTIVETo identify the causative mutation in a Chinese family affected with dentinogenesis imperfecta shields type II (DGI-II).

METHODSWith informed consent obtained from all participants, peripheral blood or chorionic villi samples were collected from the family members. Genomic DNA was extracted using a standard SDS-proteinase K-phenol/chloroform method. The whole coding region and exon/intron boundaries of the DSPP gene were amplified with polymerase chain reaction (PCR) and subjected to Sanger sequencing. To confirm the pathogenicity of the identified mutation, an Alu I recognition sequence was introduced into the mutant allele using mismatch primers by semi-nested PCR. Restriction fragment length polymorphism (RFLP) analysis was then carried out for all family members and 60 unrelated healthy controls. Meanwhile, mini-DSPP constructs were conducted to confirm the effect of the mutation in vitro.

RESULTSA splicing site mutation, c.52-1G>A, which was located upstream of exon 3, was found in all three patients and the fetus of the proband. Restriction analysis confirmed that all unaffected individuals and the 60 healthy controls did not carry the same mutation. The expression of minigene showed that the exon 3 of the DSPP gene was skipped during the transcription.

CONCLUSIONA novel pathogenic splicing-mutation c.52-1G>A has been detected in a Chinese family affected with DGI-II, which enabled prenatal diagnosis for the fetus of the proband.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child, Preschool ; Dentinogenesis Imperfecta ; genetics ; Exons ; Extracellular Matrix Proteins ; genetics ; Female ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Phosphoproteins ; genetics ; Point Mutation ; RNA Splicing ; Sialoglycoproteins ; genetics

OBJECTIVETo analyze the features of genetic mutations underlying Wilson's disease and provide prenatal and presymptomatic diagnosis.

METHODSFor 35 pedigrees affected with the disease, the exons and exon-intron boundaries of the ATP7B gene were amplified with polymerase chain reaction and subjected to Sanger sequencing. After the genotypes of parents of the probands were determined, prenatal diagnosis were performed through chorionic villus sampling.

RESULTSThe overall rate for mutation detection was 92.9%. A total of 24 distinct mutations were detected, which included 7 novel mutations, i.e., c.3871G>A(p.A1291T), c.2593_2594insGTCA, c.2790_2792delCAT, c.3661_3663delGGG, c.3700delG, c.4094_4097delCTGT, and IVS6+1G>A. Three mutations, including R778L (c.2333G>T)(45.7%), A874V (c.2621C>T)(7.1%) and P992L (c.2975C>T)(7.1%), were relatively common. Two presymptomatic patients were detected through familial screening, for whom treatment was initiated. Prenatal genetic diagnosis has verified three healthy fetuses and one carrier.

CONCLUSIONIn this study the most popular mutation ofATP7B gene is R778L and 7 novel mutations have been identified in this gene. For pedigrees of Wilson's disease, genetic counseling in addition with prenatal and presymptomatic diagnosis should be provided through Sanger sequencing and haplotype analysis.

Adenosine Triphosphatases ; genetics ; Adult ; Base Sequence ; Cation Transport Proteins ; genetics ; Copper-transporting ATPases ; DNA Mutational Analysis ; Female ; Genotype ; Hepatolenticular Degeneration ; embryology ; enzymology ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis

OBJECTIVETo determine the incidence and molecular characteristics of G6PD deficiency in Chaozhou region of eastern Guangdong Province.

METHODSG6PD enzyme activity was assayed with an auto-bioanalyzer. Reverse dot blotting (RDB) was used for detecting 6 common G6PD mutations. Samples with no mutation detected by RDB were further sequenced for unknown mutations.

RESULTSThe rate of G6PD deficiency was 3.36% (142/4224). 2.33% (47/2013) of males and 4.3% (95/2208) of females were affected. 12 mutations were detected among the 142 patients, which included c.1376G>T, c.1388G>A, c.1024C>T, c.392G>T, c.871G>A, c.95A>G, c.517T>C, c.131C>G, c.1376G>T/c.517T>C, c.871G>A/IVS-1193T>C/c.1311C>T, c.1376G>T/IVS-11, 93T>C/c.1311C>T and c.1376G>T/c.486_34delT (rs3216174).

CONCLUSIONThe incidence of G6PD deficiency in Chaozhou region was lower than that of the Hakka population of Guangdong Province, and the mutation types were diversely distributed in this region. c.1376G>T, c.1388G>A and c.1024C>T were the most common mutations, which was followed by c.517T>C. In addition, c.131C>G has been first discovered in the Chinese population. c.1376G>T/c.517T>C and c.1376G>T/c.486_34delT(rs3216174) were new types of compound heterozygous mutations in females.

Adolescent ; Base Sequence ; China ; epidemiology ; ethnology ; Female ; Genotype ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; enzymology ; epidemiology ; ethnology ; genetics ; Humans ; Incidence ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Mutation

OBJECTIVETo explore the effect of false positive signals during detection of BCR/ABL fusion gene by fluorescence in situ hybridization (FISH), and develop a method for calibration.

METHODSNormal specimens were mixed with BCR/ABL positive specimens in which presented signal pattern of 1-red-2-green-1-fusion (1R2G1F) using dual color dual fusion (DCDF) probes and 1-red-1-green-1-fusion (1R1G1F) using extra signal (ES) probes in different proportions. Mixed samples were detected using DCDF and ES probes. Results of DCDF probes, ES probe before calibration, ES probes after calibration and theoretical results were compared by binomial distribution in different proportions.

RESULTSThe rate of false positive signals has risen with increase of negative rate. A significant difference was found between theoretical proportion and results without calibration in negative level, 5%, 10% and 25% positive level (P<0.05). There was no significant difference between theoretical proportion and results without calibration in 50% and 90% positive level (P>0.05). Also there was no significant difference between theoretical proportion and calibrated results (P>0.05).

CONCLUSIONCalibration of FISH result can delimitate the effect of false positives, and can provide more reliable results in cases with low level positive rates.

Adult ; Calibration ; False Positive Reactions ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; methods ; standards ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; Young Adult

OBJECTIVETo explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.

METHODSAn eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.

RESULTSThe eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.

CONCLUSIONThe ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

Animals ; Base Sequence ; Blood Platelets ; cytology ; metabolism ; CHO Cells ; Cloning, Molecular ; Codon, Nonsense ; genetics ; Cricetinae ; Cricetulus ; Humans ; Integrin beta3 ; genetics ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; metabolism ; Point Mutation

OBJECTIVETo study the clinical features and diagnosis of 7 patients with atypical mantle cell lymphoma (MCL).

METHODSThe 7 MCL patients were misdiagnosed as chronic lymphocytic leukemia (CLL) due to a score of 4 for their immunophenotypes. The clinical features and diagnosis of such patients were retrospectively analyzed.

RESULTSSix patients had superficial lymphadenectasis but their lymph nodes could not be palpated. All 7 patients were as stage IV considering bone marrow infiltration. Scores of immunophenotype of CLL were 4, and interphase fluorescence in situ hybridization (FISH) for t(11;14) were positive in all patients.

CONCLUSIONSome MCL patients have clinical features similar to CLL. Interphase FISH can play an important role in the diagnosis of MCL.

Aged ; Chromosomes, Human, Pair 11 ; genetics ; Chromosomes, Human, Pair 14 ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Lymphoma, Mantle-Cell ; diagnosis ; genetics ; Male ; Middle Aged ; Translocation, Genetic

OBJECTIVETo assess the value of eight-probe fluorescence in situ hybridization (FISH) and R-banding karyotype analysis for the diagnosis of acute lymphoblastic leukemia (ALL).

METHODSWith the eight-probe FISH (using probes for MYC, P16, E2A, CHIC2/D10Z1/D17Z1, TEL/AMLl, MLL, BCR/ABL1, and IGH) and R-banding karyotype analysis, 237 cases of ALL were analyzed.

RESULTSCytogenetic changes were detected in 135 (56.96%) of all cases, which have involved MYC, P16, E2A, CHIC2/D10Z1/D17Z1, TEL/AMLl, MLL, BCR/ABL1, and IGH polyploidies. R-banding karyotype analysis has only detected abnormalities in 48 of such cases, in addition with 14 abnormalities missed by the FISH probes, which have given a total positive rate of 26.16%. The detection rate of the two methods has differed significantly(P<0.05).

CONCLUSIONCompared with the R-banding karyotype analysis, the eight-probe FISH is more accurate and efficient. Diagnosis of cytogenetic abnormalities for children with ALL using the combined method can provide a basis for evaluation of prognosis as well as personalized therapy.

Chromosome Aberrations ; Chromosome Banding ; methods ; Genetic Testing ; methods ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; methods ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; genetics

OBJECTIVETo investigate the clinical application of fluorescent in situ hybridization (FISH) for the differential diagnosis of myelodysplastic syndromes (MDS) and aplastic anemia (AA).

METHODSA FISH kit capable of detecting the chromosomal abnormalities related to MDS was used to analyze 94 patients who were suspected to have AA by bone marrow morphology.

RESULTSCytogenetic abnormalities were detected in 11 of the 94 patients, which included trisomy 8 (5 cases), 20q- (1 case) and -Y (1 case). There were 4 cases related to MDS, which included 3 cases of 5q-, in which 1 case carry 20q- at the same time, and 7q- (1 case). No significant difference was found between the MDS and AA groups in terms of age, sex or routine blood examination including absolute neutrophil count, hemoglobin content and platelet count.

CONCLUSIONFISH can detect certain cytogenetic abnormalities related to MDS in patients morphologically diagnosed as AA.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; diagnosis ; genetics ; Bone Marrow Cells ; cytology ; Child ; Chromosome Aberrations ; Chromosomes, Human, Pair 7 ; genetics ; Chromosomes, Human, Pair 8 ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Male ; Middle Aged ; Trisomy ; genetics

OBJECTIVETo provide preimplantation genetic diagnosis(PGD) for two couples carrying thalassemia mutations and chromosomal abnormalities.

METHODSCouple 1 were both carriers of β 41/42 thalassemia mutations, while the husband has carried a reciprocal translocation with a karyotype of 46,XY,inv(9)(p11;q13),t(11;22)(q25;q13). Couple 2 were both carriers of α (-SEA) thalassemia mutation. Their chromosome karyotypes were both normal, but had two spontaneous abortions. The couples had received 1 and 3 blastocysts respectively through in vitro fertilization(IVF) cycles. Following the biopsy, the cells underwent whole genome amplification, and the amplified DNA from each embryo was subjected to genetic testing and a 23-chromosome single nucleotide polymorphism(SNP) microarray assay.

RESULTSThe embryo of couple 1 was diagnosed as carrier of β 41/42 thalassemia with euploid chromosomes. The embryo was transferred and resulted in intrauterine pregnancy. Similarly, an embryo of couple 2 was verified as carrier of α (-SEA) thalassemia with euploid chromosomes.

CONCLUSIONPGD for aneuploidy coupled with testing for single gene disorders via trophectoderm biopsy and whole genome amplification is feasible. The approach can attain diagnosis with minimal damage with sound clinical outcome.

Adult ; Aneuploidy ; Blastocyst ; cytology ; Chromosome Aberrations ; Embryo Transfer ; Female ; Fertilization in Vitro ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pregnancy ; Preimplantation Diagnosis ; beta-Thalassemia ; diagnosis ; embryology ; genetics