Objective To develop an analyzing technique of single nucleotide polymorphism(SNP) based on non-probe real-time quantitative PCR, and to explore the relationship between SNP in human adiponectin (APM1) gene and type 2 diabetes (T2DM).Methods Design primers with specific 3′-terminal nucleotides and perform Real-time PCR using SYBR Green dye. Genotypes were determined by comparing the amplification efficiency of each pair of primer and verified by sequencing. SNPs 45 and 276 in APM1 of 20 T2DM, 24 obesity and 28 healthy persons were analyzed. Results This technique could discriminate genotype as efficient as sequencing. The genotype of APM1 at +45 site was significantly correlated with T2DM (P

OBJECTIVETo test the frequency and pattern of rhodopsin (RHO) mutations in Chinese retinitis pigmentosa (RP) patients and to evaluate their effects in the pathogenesis of RP.

METHODSGenomic DNA was extracted from peripheral blood samples of 100 Hong Kong Chinese RP patients. Sequence variants of the entire coding exons of the RHO gene were tested using PCR, conformation sensitive gel electrophoresis and DNA sequencing.

RESULTSTotally six nucleotide changes were identified, among which three were silent mutations, two missense mutations and one deletion mutation. P347L was found in one RP proband and her three children who also had RP. P327(1 bp del) was novel and detected in a late-onset RP patient of 53 years. Her 26-year-old daughter, also carrying the identified mutation, had no RP phenotypes except for the mottled retinal pigment epithelium (RPE) revealed by fundal examination. Neither of the two mutations was detected in normal controls.

CONCLUSIONTwo patients had disease-causing mutations in the RHO gene, thus RHO mutations cause about 2.0% (95% confidence interval: 0.2%-7.0%) of all RP among Chinese in Hong Kong. A highly conserved C-terminal sequence QVS(A)PA was altered due to P347L and thereby resulting in an aberrant subcellular localization of rhodopsin. Loss of all six phosphorylatable residues at the C-terminus and the highly conserved C-terminal sequence QVS(A)PA may occur because of P327(1 bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying P327(1 bp del) would be of greatest value.

Adolescent ; Adult ; Aged ; Child ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Female ; Gene Frequency ; Genetic Testing ; Humans ; Male ; Middle Aged ; Point Mutation ; Retinitis Pigmentosa ; genetics ; pathology ; prevention & control ; Rhodopsin ; genetics ; Sequence Deletion

OBJECTIVETo identify and evaluate mutations in the RP1 gene among Chinese patients with retinitis pigmentosa (RP).

METHODSLeukocyte DNA of 92 RP patients were collected in Hong Kong. Sequence changes of the entire coding region of the RP1 gene were examined using PCR, conformation sensitive gel electrophoresis and DNA sequencing.

RESULTSIn total, 1 nonsense mutation and 1 nonsense variant as well as 10 missense alterations were identified in the RP1 gene, among which, Arg677Ter was found in 1 RP patient and another nonsense variant, Arg1933Ter, was identified in 3 normal individuals and 1 patient with Stargardt's disease, suggesting its nonpathogenicity. Arg77Ter is expected to lead to large disruptions of the encoded protein.

CONCLUSIONSThe nonpathogenicity of Arg1933Ter indicates that the C-terminal 224 residues of RP1 protein may be not critical for RP1. The most C-terminal truncation previously reported was due to Tyr1053 (1-bp del) and occurred in RP patients. Thus RP can be caused by reduction in the level of the region of RP1 protein after codon 1052 but before 1933. To ascertain such a proposition, genotypes of more RP patients may reveal more RP causative mutations and more sequence alterations different than those of other ethnic groups.

Adolescent ; Adult ; Aged ; Child ; Codon, Nonsense ; DNA Mutational Analysis ; Eye Proteins ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Retinitis Pigmentosa ; genetics

OBJECTIVETo investigate the frequency and pattern of RP1 point mutations in Chinese retinitis pigmentosa (RP) patients and to examine their effects on the development of RP.

METHODSConformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing were used to determine sequence alterations occurring in the entire coding region of the RP1 gene in 101 Chinese RP patients in Hong Kong.

RESULTSR677X was detected in one RP patient. A nonpathogenic nonsense mutation, R1933X, was identified in three normal individuals and one patient with Stargardt disease. The frequency of RP1 mutations among all RP patients in this study is 1/101. R677X is expected to lead to large disruptions of the encoded protein. Additionally, 10 more missense alterations in the RP1 gene were identified in the subjects of this study. Apart from M479I whose pathogenicity can not be determined currently, other sequence changes are just polymorphisms of the RP1 gene.

CONCLUSIONThe nonpathogenicity of R1933X indicates that the C-terminal 224 residues of RP1 protein may be not critical for RP1. Recently, a C-termnal truncating mutation, Y1053(1 bp del), was reported to occur in an RP patient. Thus RP can be caused by lack of the region of RP1 protein after codon 1052 but before 1933. To confirm such a proposition, a large genotyping study is necessary and is likely to reveal more RP causative mutations and uncover more sequence alterations different from those of other ethnic groups.

Adolescent ; Adult ; Aged ; Child ; China ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Eye Proteins ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Mutation, Missense ; Retinitis Pigmentosa ; genetics

Objective:To establish autoverification rules for coagulation tests in multicenter cooperative units, in order to reduce workload for manual review of suspected results and shorten turnaround time (TAT) of test reports, while ensure the accuracy of results.Methods:A total of 14 394 blood samples were collected from fourteen hospitals during December 2019 to March 2020. These samples included: Rules Establishment Group 11 230 cases, including 1 182 cases for Delta check rules; Rules Validation Group 3 164 cases, including 487cases for Delta check; Clinical Application Trial Group 77 269 cases. Samples were analyzed for coagulation tests using Sysmex CS series automatic coagulation analyzers, and the clinical information, instrument parameters, test results, clinical diagnosis, medication history of anticoagulant and other relative results such as HCT, TG, TBIL, DBIL were summarized; on the basis of historical data, the 2.5 and 97.5 percentile of all data arranged from low to high were initially accumulated; on the basis of clinical suggestions, critical values and specific drug use as well as relative guidelines, autoverification rules and limits were established.The rules were then input into middleware, in which Stage I/Stage II validation was done. Positive coincidence, negative coincidence, false negative, false positive, autoverification pass rate, passing accuracy (coincidence of autoverification and manual verification) were calculated. Autoverification rules underwent trial application in coagulation results reports.Results:(1) The autoverification algorisms involve 33 rules regarding PT/INR, APTT, FBG, D-dimer, FDP,Delta check, reaction curve and sample abnormalities; (2)Autoverification Establishment Group showed autoverification pass rate was 68.42% (7 684/11 230), the false negative rate was 0%(0/11230), coincidence of autoverification and manual verification was 98.51%(11 063/11 230), in which positive coincidence and negative coincidence were respectively 30.09% (3 379/11 230) and 68.42%(7 684/11 230); Autoverification Validation Group showed autoverification pass rate was 60.37%(1 910/3 164), the false negative rate was 0%(0/11 230), coincidence of autoverification and manual verification was 97.79%(3 094/3 164), in which positive coincidence and negative coincidence were respectively 37.42%(1 184/3 164) and 60.37%(1 910/3 164); (3) Trialed implementation of these autoverification rules on 77 269 coagulation samples showed that the average TAT shortened by 8.5 min-83.1 min.Conclusions:This study established 33 autoverification rules in coagulation tests. Validation showedthese rules could ensure test quality while shortening TAT and lighten manual workload.