Objective:To analyze the clinical features and mutation of myeloid differentiation factor 88 (MYD88) L265P in patients with diffuse large B-cell lymphoma (DLBCL) of central nervous system (CNS).Methods:The clinicopathological materials of 45 cases of DLBCL of CNS were retrospectively collected in Xuanwu Hospital, Capital Medical University from September 2014 to February 2017. The clinicopathological data were retrospectively analyzed, combined with immunohistochemistry, EB virus in situ hybridization, imaging and medical history. The mutation of MYD88 L265P gene was detected by pyrosequencing and its clinical significance was analyzed. Results:The age of the patients ranged from 42 to 82 years [(57.6±8.8) years], including 24 males and 21 females. Totally 93.3% (42/45) of the patients had supratentorial tumours, which were single or multiple. The cerebral hemisphere (31/45, 68.9%) was the most common involved site, and 21 cases (21/45, 46.7%) had multiple lesions. Histologically, DLBCL in the CNS showed diffuse infiltration of tumor tissue, some of which grew around blood vessels in a "sleeve" arrangement. CD 20 and CD 79a were diffusely and strongly positive. Thirty-nine cases (39/45, 86.7%) were non-germinal center B cell (non-GCB) subtype and 6 cases (6/45, 13.3%) were germinal center B cell (GCB) subtype. MYD88 L265P mutation was found in 64.4% (29/45) patients. There was statistically significant difference between non-GCB type (71.8%, 28/39) and GCB type DLBCL (1/6, P=0.017). Compared with the operation/biopsy group without chemotherapy, operation+chemotherapy, biopsy+chemotherapy, operation/biopsy+chemotherapy+stem cell transplantation can improve the survival and prognosis ( HR=0.05, 95% CI 0.01-0.33 , P=0.002; HR=0.04, 95% CI 0.01-0.36 , P=0.004; HR=0.01, 95% CI 0.00-0.17 , P=0.001; respectively). Conclusions:DLBCL of the CNS is aggressive tumor with poor prognosis, the clinical manifestations are complex and diverse, and the diagnosis is challenging. MYD88 L265P is a common and specific gene mutation in primary CNS lymphoma(PCNSL), which is of great significance in the diagnosis and treatment of lymphoma. The MYD88 L265P mutation was more frequently detected in non-GCB than GCB subtype. Chemotherapy can improve the survival rate of PCNSL patients. If chemotherapy achieves complete remission and autologous hematopoietic stem cell transplantation is performed, there may be a chance of long-term survival.

Objective:To analyze the clinicopathological features of chordoid glioma.Methods:A total of 12 cases of chordoid gliomas from 2009 to 2020 in Xuanwu Hospital of Capital Medical University and General Hospital of Chinese People′s Liberation Army were retrospectively analyzed. The clinical and imaging characteristics, pathologic and molecular characteristics were analyzed, and the relevant literature was reviewed.Results:All 12 patients (4 males and 8 females) aged from 25 to 67 years (mean 39 years) and mainly had a history of headache or/and vision loss. MRI showed that the lesions located in the third ventricle, and they showed abnormal enhancement. Pathologically, these 12 cases displayed the morphologic characteristics of chordoid gliomas, including papillary structures in two cases. Immunohistochemically, GFAP and vimentin were expressed in all 12 cases (12/12). TTF1 was also expressed in all cases (10/10). CD34 and CKpan were seen in 11 cases (11/12). EMA with dot-and/or-ring like positivity was seen in 9 cases (9/10). Tissues were available in nine chordoid gliomas for Sanger sequencing to detect PRKCA and IDH gene mutation, and eight cases (8/9) showed PRKCA gene D463H mutation. None of these cases showed IDH1 R132 and IDH2 R172 mutation. All 12 patients underwent surgery, and four were lost to follow up. The remaining eight patients were progression or recurrence free at last follow-up in January 2021.Conclusions:Chordoid gliomas have relatively distinguishing clinical and histopathological features. PRKCA gene mutation in chordoid gliomas can be considered as a biomarker for the diagnosis and differential diagnosis of chordoid gliomas, and may provide a direction for future targeted therapy.

BACKGROUND: The special genetic law of short tandem repeat on chromosome X (X-STR) makes it incomparable with autosome markers in forensic identification. However, the population genetics data is far less than that of the autosome STR, and especially the haplotype data are rarely reported.OBJECTIVE: To study the genetic polymorphism of 12 X-STR loci in Shenzhen area by pedigree analysis, aiming to provide scientific and effective data for the application of X-STR in forensic medicine and genetics. METHODS: The blood samples of 118 families were taken to extract DNA by Chelex-100, followed by PCR amplification using Investigator Argus X-12 kit. The frequency of alleles of 231 unrelated individuals was counted by direct counting method and Excel software. Hardy-Weinberg equilibrium test was performed on 12 X-STR loci of female samples by chi-square test. Discrimination power and mean exclusion chance were calculated according to the formula. Pedigree analysis was done to identify haplotypes of female samples and the haplotype frequencies of 4 linkage groups in 111 fathers and 119 mothers were calculated using direct counting method and Excel software.RESULTS AND CONCLUSION: In this study, 349 haplotypes were obtained. There were 238, 139, 153 and 157 haplotypes in linkage groups X1-X4, respectively. The polymorphism of DXS10135 locus was the highest with 21 alleles,while the polymorphism of DXS7423 locus was the worst with only 4 alleles. The combined discrimination power was 0.99999999 in males and 0.99999999 in females. The combined mean exclusion chance was 0.99999999 in trio cases,and 0.99999811 in duo cases. These findings indicate that the X-12 detection system has high polymorphism in Shenzhen Han population, and has important application value in forensic individual identification and paternity testing.

Objective To analysis the genomic sequence of a novel human leukocyte antigen (HLA)-B*3818 allele.Methods Full length genomic sequence of an unknown HLA-B allele was cloned,followed by bi-directional sequencing and the specificity of the antigen coded by this novel allele was defined by microcytotoxicity assay.The frequency and haplotype of this novel allele was acquired by population census and parentage analysis.Results The full length genomic sequence of this novel HLA-B*3818 allele with accession number FJ561482 differs from HLA-B*380201 by two nucleotide changes in exon 4 and intron 5,respectively.One change is located at nt 660 in exon 4 where C→A alternation,which results in an amino acid substitution from Asp(GAC)to Glu(GAA)at codon 196.This alternation is a new single nucleotide polymorphism compared with all other HLA-B alleles.Another is located at genomic position 2133 in intron 5(A→C).Except for this substitution,the intron sequences of HLA-B*3818 allele are identical to those of other HLA-B*38 alleles including HLA-B*380101,B*380201 and B*3814.The serological specificity of HLA-B*3818 is B38 and the frequency of this new allele is less than 0.000 5 in Chinese Han population.The parentage analysis showed the haplotype of novel allele is A*030101-Cw*010201-B*3818-DRB1*1312-DOB1*060101.Conclusion The simultaneous mutations in exon and intron were found in the Hovel HLA-B*3818 allele,and so it can present more sequence information for studies and applications associated with HIA genes by analyzing the genomic sequences of novel HLA alleles.

Objective To evaluate the heterozygous ambiguity resolution primers (HARPs) method in resolving ambiguous genotyping results of human leukocyte antigen (HLA) genes in Chinese Hart population, and choose some appropriate HARPs primers. Methods HLA-A, HLA-B and HLA-DRB1 genes of 416 southern Chinese Han individuals were genotyped by sequence-based-typing(SBT) method and then the ambiguous genotyping samples were sequenced again by HARPs primers provided by American Atria company. Results The percentage of ambiguous genotyping samples resolved by HARPs for HLA-A, HLA-B and HLA-DRBI locus was 86.3% (132/153), 73.9% (130/176) and 38.1% (85/223) respectively. Among them, 48.5% (64/132)HLA-A, 80.0% (104/130)HLA-B and all HLA-DRB1(85/85)samples only need one primer, 47.7 % (63/132)HLA-A and 20.0% (26/130)HLA-B samples need two primers. Three to six different HARPs primers can resolve more than 90% ambiguities. Conclusion HARPs is a convenient method and could be a routine method to resolve ambiguities for HLA-A, HLA-B and HLA-DRB1 genes genotyped by SBT in Chinese Han population.

AIM: To identify human leukocyte antigen (HLA)-DRB11454 allele and HLA-DRB1 exon 3 sequence information in the Chinese population, which is significant for organ transplantation, cell transplantation, and human genetics.METHODS: Polymerase chain reaction sequence-based typing (PCR-SBT) was used to identify HLA-DRB1 alleles from 58 donor-recipient individuals who would undergo haemopoietic stem cell transplantation. Medium to high resolution polymerase chain reaction-reverse sequence specific oligonucleotide probe (PCR-RSSOP) was used to identify HLA-DRB1 alleles from 1 268 healthy donors from Guangdong province. The some ambiguous results of HLA-DRB114-associated alleles were confirmed by high resolution polymerase chain reaction-sequence-specific primer typing (PCR-SSP).RESULTS: HLA-DRB11403, 1406, 1410, 1412, 1418, 1425 and 1454 alleles were detected in 1 268 healthy donors.HLA-DRB11454 was confirmed in 8 ambiguous results of HLA-DRB11401/1434/1454 alleles, and HLA-DRB11454 was one of common alleles of HLA-DRB114 allele group in Guangdong population. HLA-DRB114 exon 3 sequence information was confirmed to be polymorphic in Chinese population.CONCLUSION: HLA-DRB11454 and exon 3 of DRB1 are confirmed to be polymorphic in Chinese population, further elucidating that HLA-DRB1 axon 3 sequence information is important for Han population and some minority groups.

Objective To study the distributive characteristics of HLA-A,B,DRBI alleles and haplotypes patients with ALL in southern Chinese Han.Methods The frequencies of HLA-A,B,DRB1alleles and haplotypes were estimated by Expectation-Maximization method based on the genotypes of 572patients with ALL and 5645 unrelated health donors,and then compared by chi-square test.Results The frequencies of HLA-A33(7.15%vs 9.3%,OR=0.73,P＜0.05),B58(5.93%vs 8.75%,OR=0.64,P＜0.05),DRB1*17(5.15%vs 6.30%,OR=0.82,P＜0.05)alleles and HLA-A33-B58-DRB1*17(2.46%vs 4.14%,OB=0.35,P＜0.05)haplotype were significantly lower in ALL patient groups than that in controls.The frequencies of HLA-A3(2.1%vs 1.26%,OR=1.7,P＜0.05),B51(7.25%vs 5.78%,OR=1.3,P＜0.05)and DRB*12 (16.13%vs 12.99%,OR=1.35,P＜0.05)alleles and A2-B51-DRB1*12(1.24%vs.0.89%,OR=1.66,P＜0.05)haplotype were significantly higher in ALL patient groups than that in controls.Conclusion These results indieated that HLA-A33-B58-DRB1*17 haplotype was a associated with a diminished incidence of ALL.and HLA-A3 auele or A2-B51-DRB1*12 haplotype was weakly associated with ALL.

BACKGROUND: Umbilical cord blood (UCB) with limited karyocytes is mainly used in child patients. Recently, physicians have tried to mix two units of cord blood in the treatment of adults with hematological system diseases.OBJECTIVE: To monitor quantitatively the dynamic changes and the development rules of engraftment, chimera types and relative amount after allogeneic transplantation of mixed UCB from two units in adults with leukemia.DESIGN: Donors and the recipient were regarded as observational subjects in umbilical cord blood transplantation (UCBT). DNA extracted from blood samples of donors and the recipient before and after transplantation was considered as detecting samples. Short tandem repeat (STR) loci were as observational measures.SETTING: Key Laboratory of Immunology and Genetics of Institute of Transfusion Medicine of Shenzhen Blood Center.PARTICIPANT: A 43-year male patient with acute myeloid leukemia (AML), 75 kg, who was hospitalized at Shenzhen Hospital of Peking University, was enrolled in June 2005. The patient received two units of human leucocyte antigen (HLA), one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2) at month 6 after complete remission from first chemotherapy. UCB was collected from Guangzhou umbilical cord blood bank. The patient signed the informed consent.METHODS: The adult with AML received two units of HLA, one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2). Nine STR loci of the blood sample were determined before and after transplantation by quantitative technique of fluorescence labeling with multiplex polymerase chain reaction (MPCR), while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. The relative amount of two units of UCB was calculated in the patient after transplantation according to the differential gene peak areas of two donors with 377XL DNA sequencer after fluorescence scanning. The engraftment level and the development rules of donors' cells were analyzed quantitatively. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment.MAIN OUTCOME MEASURES: After UCBT, transition process of nine STR loci of the recipient and two donors was observed, and engraftment was quantitatively and qualitatively described.RESULTS: Two units of UCB at day 15 after transplantation were engrafted simultaneously and revealed a complete chimera of the two. The relative amounts of UCB 1 and UCB 2 were 51.3% and 48.7%, respectively. Subsequently, UCB 1 went up to 70.0% and UCB 2 declined to 30.0% at day 30. However, only the genotype of UCB 1 was detected at day 52, and engraftment turned to a complete chimera of a single donor. The one with fewer karyocytes was rejected and the one with more karyocytes was engrafted for a long term.CONCLUSION: To detect quantitatively STR chimera with fluorescence labeling and MPCR can show precisely the engraftment level and the change of two units of UCB. It provides an accurate and reliable experimental basis for clinical UCB application and donor selection. It is proved that adult transplantation at the same time with mixed UCB from two units HLA one locus mismatched unrelated donors is feasible.

BACKGROUND: The judgment of the engraftment of hematopoietic stem cells after transplantation mainly depends on various genetic labeling in vivo, which are different in sensitivity and effectiveness, thus a method with powerful differential ability, high sensitivity and not restricted by sex is to be established.OBJECTIVE: To observe the DNA genetic loci of short tandem repeat in the blood samples of both donors and recipients before allo-hematopoietic stem cell transplantation and those of recipients at different time points after transplantation.DESIGN: An observation measurement.SETTING: Laboratory of Immunogenetics, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center.PARTICIPANTS: Blood samples of 18 pairs of donors and recipients, who were successfully matched and accepted hematopoietic stem cell transplantation, were selected from the Laboratory of Immunogenetics, Shenzhen Institute of Transfusion Medicine, Shenzhen Blood Center from February 2004 to December 2005. Among the 18 patients, there were 10 males and 8 females, with a mean age of 35 years old, including 6 cases of them were donated by relatives with blood relationship, and 12 cases by volunteers without blood relationship. Informed consents were obtained from all the participants.METHODS: The blood samples of both donors and recipients before transplantation and the blood samples of recipients after transplantation were collected, and the fluorescence labeling short tandem repeat technique was used to detect the 15 loci for short tandem repeat and Amelogenin sex locus, so that the differential loci between the donor and recipient could be screened. The engraftment and dynamic changes of the short tandem repeat genes of the donors in the recipients after transplantation were observed, the times for the earliest occurrences of short tandem repeat genes of the donors and the complete chimerism were recorded.MAIN OUTCOME MEASURES: ① Differential genes between the donors and recipients before transplantation;②Times for the earliest occurrences of short tandem repeat genes of the donors and the complete chimerism.RESULTS: All the 18 pairs of donors and recipients were involved in the final analysis of results. Satisfactory results of the typing at the 15 loci for short tandem repeat and 1 sex locus in the 18 pairs of samples of both donors and recipients before transplantation and the sample of the recipients after transplantation respectively. Averagely 12.4 (8-15) differential loci for short tandem repeat could be distinguished between the donors and recipients. ②After transplantation, short tandem repeat genes could be detected the earliest at 8 (5-14) days averagely, It took 14 (9-23) days averagely for short tandem repeat loci to convert from recipient type completely into donor type, and the engraftment converted from the recipient chimerism types completely into the donor types.CONCLUSION: The fluorescence labeling compound amplification of short tandem repeat technique can precisely measure the number of PCR products, describe the engraftment of hematopoietic stem cells and the whole process of development. It can also provide accurate and timely information for the early judgement of engraftment, predicting failure of transplantation and controlling recurrence.

0.05);but as comparing with high dose group there is obvious significance(P