Objective To develop a multiplex sequence-specific PCR assay for simultaneous screening of 5 types of JAK2 mutations and investigate its clinical application value. Methods Multiplex sequence-specific PCR assay for simultaneous screening of JAK2 V617F, K539L (include 2 types of gene mutations), N542-E543del and E543-D544del mutations were developed. 115 patients with myeloproliferative diseases (MPD) including 61 polycythemia vera (PV) cases, 43 essential thrombocythemia (ET) cases and 11 primary myelofibrosis (MF) cases were analyzed. Results The assay can screen the 5 types of JAK2 mutations efficiently. The detection sensitivity is 1% for JAK2 V617F mutation and 0.1% for the other mutations. JAK2 V617F mutation and JAK2 exon12 mutation were detected in 56 and 3 of the 61 PV samples, respectively. 27 of the 43 ET samples and 6 of the 11 MF samples were JAK2 V617F positive, but no JAK2 exon12 mutation was found in both groups. The 3 cases carrying JAK2 exon12 mutation had the clinical feature of erythrocytosis and erythropoietin-independent erythroid colony formation but without apparent leukocytosis, thrombocytosis and splenectasis. Conclusion The assay can simultaneously screen 5 types of JAK2 mutations with high sensitivity and thus lead to an increased detection rate.

Objective To analyze the etiological factor and genetic feature of a familial hemophagocytic lymphohistiocytosis patient with PRF1 mutation (FHL2) with human herpesvirus 7 (HHV7)infection and its family constellation. Methods Clinical characteristics, laboratory examinations of a FHL2 case with HHV7 infection were reported. HHV1-HHV8 virus DNA was screened by PCR; NK cell function was analyzed by flow cytometry; PRF1 gene mutations were analyzed by PCR and direct sequencing, structure of mutant PRF1 proteins were analyzed using ExPasy and I-TASSER server and genetics pedigree were analyzed. Results The patient's HHV7 viral was detected positive with DNA copy number of 350/106 peripheral nucleated cells. Flow cytometry analysis showed decrease both in proportion of perforin positive NK cells and perforin protein expression. Genetic testing showed PRF1 biallelic heterozygote mutations (c. 503G ＞ A/p. S168N and c. 1177T ＞ C/p. C393R) and pedigree analysis showed they were inherited. The patient was then treated with antivirus therapy, dexamethasone and VP16 therapy, but only achieved partial response. The patient was then followed by human leukocyte antigen 10/10 allele identical nonconsanguinity allogeneic hematopoietic stem cell transplantations (allo-HSCT) and soon the successful implantation of donor hematopoietic cells and persistent recovery was achieved. The patient was now surviving without recurrence for 9 months after allo-HSCT. Conclusions FHL is prone to be misdiagnosed as lymphoma. Genetic analysis of related gene mutation and herpes simplex virus detection will help in early and accurate diagnosis. Allo-HSCT is a fundamental treatment of FHL.

Objective: To investigate genotypes and drug susceptibility of the 100 strains of Candida glabrata isolated from 100 women (including 50 pregnant women) in order to study the drug-resistance and gene polymorphism, and to investigate the correlation of drug-resistance, gestation and gene polymorphism. Methods: Multilocus sequence typing (MLST) technique was introduced to identify sequences of 6 housekeeping genes from 100 isolates of Candida glabrata. The results were compared with sequence information in MLST databases by Clustalx software to determine a strain allelic profile and sequence type (ST). Drawing the phylogenetic tree by weighted paired group average method and the minimal spanning tree method of MEGA6.0 software, the microevolution and relationship between different strains were analyzed. ATB FUNGUS semi-automatic system was used to test the drug susceptibility. Fisher analysis method was used to analyze the correlation between the genotypes and pregnancy.Ridit analysis method was used to analyze the correlation between the genotypes and drug susceptibility. Results: The 100 isolates belonged to 34 clone sequences. There were 53 isolates belonged to ST-7 ( 26 isolates of pregnancy), 7 isolates belonged to ST-3 (3 isolates of pregnancy), 6 isolates belonged to ST-19 (5 isolates of pregnancy ), 3 isolates belonged to ST-15 (1 isolate of pregnancy), 2 isolates belonged to ST-10 (2 isolates of pregnancy), 1 isolate belonged to the other types of ST. Total of 100 isolates of Candida glabrata were 100% sensitive to fluorocytosine and amphotericin. The effect of itraconazole was poor with the sensitive rate of 20%. The resistance rates of fluconazole and voriconazole were 4% and 1% respectively. All genotypes were sensitive to voriconazole except ST-X1. In the correlation between genotype and itraconazole resistance, ST-7 as the standard group, the Ridit values in the group of ST-15, ST-19 and other types of ST were not included the mean Ridit value of the standard group in itraconazole (0.5). The system evolution tree was built using the neighbor-joining method (NJ) . All genotypes could be divided into 3 groups, as Ⅰ, Ⅱ, Ⅲ. Group Ⅰ had 44 cases, group Ⅱ had 53 cases , group Ⅲ had 3 cases . All the collected clinical strains had small genetic distances during molecular evolution. Conclusions ST-7 was the dominant genotype in Guiyang. No correlation between different STs and patients′ pregnancy was found. The different drug susceptibility in itraconazole between ST-7, ST-15, ST-19 and other STs were found. The Candida glabrata associated with VVC showed highly discrimative diversity. However, the phylogenic analysis exhibited genetic similarity among the strains studied.(Chin J Lab Med, 2018, 41: 596-600)

Objective To study the type and corresponding clinical characteristics of primary hemophagocytic lymphohistocytosis (HLH) associated immune gene mutations in the refractory virus infection or HLH of unknown causes. Methods From December 2009 to July 2010, the patients with refractory virus infection or HLH of unknown causes were screened for the primary HLH associated immune genes mutations by DNA sequence analysis, including PRF1, UNC13D, STX11, STXBP2, SH2D1A and XIAP. The clinical characteristics and outcomes were followed up. Results Totally 25 patients with refractory virus infection or HLH of unknown causes were investigated for the 6 genes and 13 cases were found carrying gene mutations, composing of 6 of PRF1 mutation, 3 of UNC13D, and each one of STX11,XIAP, SH2D1A and STXBP2, respectively. Among the 13 cases with gene mutations, 5 suffered from Epstein-Barr virus associated HLH( EBV-HLH), 1 human herpes virus 7 associated HLH (HHV7-HLH),1 HLH without causes, 4 chronic activated EB virus infection (CAEBV) with 1 progressing to Hodgkin's lymphoma carrying abnormal chromosome of t ( 15; 17 ) (q22; q25 ) and hyperdiploid, 2 EBV associated lymphoma. Among the other 12 patients without gene mutation, 4 suffered from EBV-HLH with 1 progressing to peripheral T lymphoma, 8 suffered from CAEBV. Conclusions Primary HLH associated immune gene mutations are critical causes of refractory virus infection of unknown causes, most patients manifest as HLH,some cases appear in CAEBV and EBV associated lymphoma. DNA sequence analysis is helpful to early diagnosis and correct decision-making for treatment.

ObjectiveTo examine whether DNA extracted from free edge fingernails specimens from patient after hematopoietic stem cell transplantation (allo-HSCT) could be used for short tandem repeat (STR) genotyping and chimerism analyzing,and to observe the chimerism status in fingernails after allo-HSCT.MethodsPeripheral blood,bone marrow,oral mucosa and free edge fingernail specimens were collected from 25 patients which allo-HSCT were performed in Beijing Dao-pei Hospital during Jul.2009 to Sep.2011 and their donor.Genomic DNA was extracted and 15 STR loci genotyping and chimerism analysis were performed.For the first group which including 12 patients,pairs of fingernail and oral mucosa specimens were collected within one month after allo-HSCT and were comparative analyzed.For the second group which including 13 patients,chimerism status in fingernail samples were analyzed 3 months or longer after allo-HSCT,and 3 patients underwent repeated testing at different times.ResultsFor the first group,4 oral mucosa specimens showed donor chimerism with varying degrees,but no donor chimerism was detected.in all of 12 fingernail specimens.For the second group,6.7％ to 82.6％ donor chimerism was detected in fingernail specimens in 5 out of 13 patients.For the 3 patients underwent repeated testing,donor chimerism was continued negative in one cases,but continued positive in the other 2 cases.ConclusionsFree edge fingernail samples of patients within one month after allo-HSCT can be used for STR typing and chimerism analysis,and it is better than oral mucosa samples.There are cells in allo-HSCT donor graft can differentiate into skin cells,donor derived skin cells chimerism can be formed and persist in some patients.Med,2012,35:23-26)

Objective To evaluate the clinical effect of fire needle in treating vitiligo and the characteristics of vitiligo image by the confocal laser scanning microscope(CLSM).Methods The randomized self-controlled experiment design was adopted.Each patient selected two symmetric or adjacent white patches and randomly received the fire needle treatment or tacrolimus treatment. The duration of treatment was 3 months.The CLSM images of white patches were recorded before treatment and after 3,6 times of fire needle treatment.Results Among 41 cases of stable stage vitiligo,The effective rates of the fire needle group and tacrolimus group were 82.9% and 78.0% respectively,and the difference was not statistically significant(P>0.05).After fire needle treat-ment,dentritic melanin cells appeared,the pigment granules gradually appeared around the basal layer and corpora papillare,and formed the pigment ring.Conclusion Fire needle and tacrolimus have the similar effect in treating vitiligo,moreover CLSM can be used as the non-invasive,objective and reliable detection means of the recovery of vitiligo melanocyte.