The study of variant s antigen expression revealing a novel c.160C>T (p.Arg54Cys) variant on GYPB*s allele associated with partial s phenotype.

BACKGROUND: Little s antigen is mainly defined by a single nucleotide polymorphism at c.143C (p.Thr48) on the GYPB gene. Several variants on GYPB can alter the expression of s antigen. The aim of this study was to investigate the molecular basis of variant s antigen expression in the Chinese population. STUDY DESIGN AND METHODS: A total of 4983 whole blood samples were collected to screen the individuals with discrepant s typing results using two different monoclonal anti-s. Then, the sequence of GYPB exon 4 was analyzed by Sanger sequencing. Flow cytometry analysis was performed to quantify s antigen expression on red blood cells (RBCs). In vitro expression study was performed to verify the effect of the GYPB variants identified on the expression of s antigen. RESULTS: Four donors were identified to have discrepant s typing results. Sanger sequencing showed that three donors carried the c.173C > G variant (p.Pro58Arg) specific for sD antigen, the other one carried a novel GYPB (c.160C > T, p.Arg54Cys) variant. Flow cytometry identified a partial and weak expression of s antigen on the RBCs of the four donors. Furthermore, in vitro expression study confirmed the effect of the two variants on the s antigen expression. CONCLUSION: The results demonstrated that in addition to p.Thr48, the two extra amino acids p.Arg54 and p.Pro58 are also important for full expression of s antigen. Since the individuals with partial s antigen are at risk for the development of alloanti-s, it is important to select at least two different monoclonal anti-s for correct s typing.

A novel FUT1*c.889C>T allele associated with the para-Bombay AB phenotype and computational evaluation of the mutation effect.

BACKGROUND: Mutation in the FUT1 gene can impact the structure and function of α-(1,2)-fucosyltransferase 1 (α2FucT1). To explain the para-Bombay phenotype of a novel FUT1 allele, three-dimensional (3D) modeling and mutation effect analysis of α2FucT1 were performed by bioinformatic tools. MATERIALS AND METHODS: Blood and saliva samples were collected from a patient who was suspected to be a para-Bombay phenotype. H, A, and B antigens were determined with routine serologic methods for those samples. FUT1 and FUT2 coding regions were determined by Sanger sequencing. The novel heterozygous mutation was confirmed by cloning and sequencing. 3D model of mutant α2FucT1 was built by Phyre 2 and the mutation effect was evaluated by Chimera, PROVEAN, and Polyphen-2. RESULTS: Weak H, A, and B antigens were detected on RBCs of the proband and normal quantities of H, A, and B antigens were observed in his saliva. Cloning sequencing showed that the proband carried a novel FUT1 allele (c.889C>T, p.Leu297Phe) and a null FUT1*01N.06 allele. 3D model showed that the p.Leu297Phe variant in α2FucT1 reduced the number of hydrogen bonds and the mutation effect was predicted to be deleterious and possibly damaging, which suggested that the conformation and activity of the enzyme might be significantly damaged. CONCLUSION: A novel missense mutation led to an amino acid variant p.Leu297Phe in α2FucT1, which was a potential cause of the inactivation of the enzyme. Computational evaluation was a convenient and useful approach for the mutation effect analysis of the enzyme.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

Molecular and serological analysis of the D variant in the Chinese population and identification of seven novel RHD alleles.

BACKGROUND: The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary. STUDY DESIGN AND METHODS: Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed. RESULTS: DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women. DISCUSSION: The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.

Secondary alloanti-D immunization post transfusion of "Asia type" DEL red blood cells.

BACKGROUND: "Asia type" DEL red blood cells (RBCs) express a very weak D antigen and cannot be detected by routine RhD typing. Thus, it is routinely typed as D-negative (D-) blood group and transfused to D- recipients. Here we described a case of secondary alloanti-D immunization that was associated with transfusion of DEL RBCs to D- recipients and was initially considered as primary alloanti-D immunization. CASE PRESENTATION: A 44-year-old D- woman (G2P2) with adenomyosis and anemia underwent transabdominal hysterectomy. She received four units of D- RBCs before operation. Before transfusion, the alloantibody screening test was negative. Four days after the first transfusion, she needed another RBC transfusion. Unexpectedly, the routine pre-transfusion alloantibody screening test became positive and anti-D (titer, 128-fold) was identified, indicating an alloanti-D immunization. The anti-D developed four days after the first transfusion was unexplained, so alloantibody identification was performed on the sample collected before the first transfusion, and weak anti-D combined with anti-E, which was not detectable during the previous routine pre-transfusion alloantibody screening test with non-enzyme-treated screening cells, was identified using bromelain-treated panel cells. The remaining blood samples of first transfusion in bag tails from two donors were collected for RHD genotyping analysis. One donor was later identified as "Asia type" DEL having RHD* 1227 A/01 N.01 genotype. CONCLUSION: Caution should be applied when we conclude that transfusion of "Asia type" DEL RBCs to true D- recipients could induce primary alloanti-D immunization, especially if the short time interval between transfusion and detection of anti-D is observed.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

BACKGROUND: The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed. STUDY DESIGN AND METHODS: DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast. RESULTS: The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals. CONCLUSION: The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Serological screening and genetic analysis of RhCE variants in the Chinese Southern Han donors.

OBJECTIVES: To screen RhCE variants in the Chinese Southern Han donors for molecular genetic analysis. BACKGROUND: More than hundreds of RhCE variant alleles have been described to resulting in weak and/or partial expression of RhCE antigens, generation of low-prevalence antigens and/or absence of a high-prevalence antigen of Rh system, which mainly reported in the people of African origin. In this study, the serological screening and molecular genetic analysis of RhCE variants were performed in the Chinese Southern Han donors. METHODS: The blood samples of E(+) donors were preliminarily collected. Then, RhCE antigens of the E(+) samples were further typed by using two sets of monoclonal anti-C, anti-c, anti-e and another anti-E. When weak expression of RhCE antigens was found, direct sequencing for 10 exons of RHCE gene, RH genotyping analysis by using multiplex ligation-dependent probe amplification, flow cytometric analysis and even cDNA sequencing were performed. RESULTS: A total of 4487 E(+) samples were collected and four samples with weak expression of antigens were detected. RHCE*Ce375G and RHCE*Ce667T variant alleles were identified in two samples with weak expression of e antigen, respectively. But no variant alleles were found in another two samples with weak expression of C antigen. CONCLUSION: The variant RHCE*Ce375G validated by mRNA sequencing and the deduced RHCE*Ce667T alleles were firstly identified in the Chinese population. The DCE haplotype might account for the weak expression of C antigen in two donors.

Identification of a novel DI*02(2558T) allele associated with weakened expression of DI2 antigen.

BACKGROUND: The distribution of DI1/DI2 antigens of the Diego blood group system is polymorphic in Mongoloid populations and the corresponding alloantibodies are clinically significant. Here a novel DI variant was found by donor screening, and the effect of the novel and previously reported mutations on expression of DI1/DI2 antigens and Band 3 protein was explored. STUDY DESIGN AND METHODS: DNA samples of 1150 Chinese donors were collected. DI*01/DI*02 genotyping was determined by Sanger sequencing. For the carrier of novel allele, the expression of Band 3 and DI1/DI2 antigens on red blood cells (RBCs) was detected by Western blot and flow cytometry, respectively. in vitro expression studies were conducted by transfecting the mutant (including the novel and three reported DI*02(2534T), DI*02(2358_2359insCAC), and DI*02(2572T) alleles) or wild-type DI*02 constructs into HEK 293T cells, the expression of Band 3 and DI1/DI2 antigens was analyzed. RESULTS: A novel heterozygous mutation (c.2558C>T, p.Thr853Met), which is located near the DI1/DI2 polymorphism site (c.2561T>C), was identified in a donor with DI:-1,2 phenotype. Reduced expression of DI2 antigen was observed on the RBCs, while weakened expression of Band 3 and absence of DI2 antigen were detected in cells transfected with the mutant DI*02(2558T) construct. In addition, absent or decreased expression of Band 3 and DI2 antigen was also detected in cells transfected with three reported mutant constructs. CONCLUSION: The novel DI*02(2558T) allele and three previously described DI mutations can affect the expression of Band 3 protein and/or DI2 antigen and/or interfere with DI*01/DI*02 genotyping result.

A variant RhAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes.

BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c- E- e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.

Genotyping analysis of MNS blood group GP(B-A-B) hybrid glycophorins in the Chinese Southern Han population using a high-resolution melting assay.

BACKGROUND: MNS hybrid GP(B-A-B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B-A-B) hybrid glycophorins based on high-resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population. STUDY DESIGN AND METHODS: DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B-A-B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation-dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles. RESULTS: A total of five GYP(B-A-B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B-A-B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild-type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5' inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single-nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles. CONCLUSION: The HRM assay could distinguish the GYP(B-A-B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B-A-B) genotyping kits for the Chinese population.

[Phenotype Types and Genetic Mutation Mechanism of Rhesus D Variant Individuals].

OBJECTIVE: To explore the phenotype types and genetic mutation mechanism of Rhesus D variant individuals. METHODS: Fouty-eight peripheral blood samples of pregnancies and blood donors who had been identified as Rhesus D variant by using routine serologic methods were collected from January 2013 to October 2015 in our center. The multiple ligation-dependent probe amplification(MLPA) was used to determine the RHD after genomic DNA had been extracted from the blood sample, then the data including gene copy number variations, point mutations, deletions and hybrid fusions were analyzed by GeneMarker software. All exons of blood sample RHD were amplified via PCR and analyzed by sequencing when its MLPA results were not in accordance with serologic results. Cloning and haplotype sequencing were performed if novel allele had been found. RESULTS: Rh phenotypes of the 48 samples were typed as following: 20 cases out of 48 were CcDee(41.7%, 20/48),12 cases were ccDEe (25%,12/48), 11 cases were CCDee(22.9%, 11/48), 5 cases were CcDEe (10.4%, 5/48), respectively. The MLPA analysis showed that 38 cases possessed only 1 variant allele(RHD zygosity was Dvd), while 10 cases possessed 2 variant alleles(RHD zygosity was DvDv). In Dvd type individuals, point mutations were found in 18 cases and RHD/CE hybrid fusions were found in 20 cases. In DvDv individuals, point mutations combined with RHD/CE hybrid fusions were found in 9 cases, deletion combined with RHD/CE hybrid fusions were found in 1 case. Variant alleles analysis basing on MLPA showed that 14 cases were weak D 15 and 22 cases were RhD VI type 3, however, the variant alleles were not identified in 7 cases due to lack of detecting probes and were identified via sequencing analysis. Two novel mutations, 79-81delCTC and 689G>A were also certificated by sequencing in 2 cases. CONCLUSION: CcDee is the major Rh phenotype in RhD variants, weak D 15 and RhD VI type 3 are the main serologic type of RhD variants, point mutation and RHD/CE hybrid fusions are main molecular mechanism for RhD variant phenotype. Besides, 79-81delCTC and 689G>A are two novel alleles.

Validation of the multiplex ligation-dependent probe amplification assay and its application on the distribution study of the major alleles of 17 blood group systems in Chinese donors from Guangzhou.

BACKGROUND: Genotyping platforms for common red blood cell (RBC) antigens have been successfully applied in Caucasian and black populations but not in Chinese populations. In this study, a genotyping assay based on multiplex ligation-dependent probe amplification (MLPA) technology was applied in a Chinese population to validate the MLPA probes. Subsequently, the comprehensive distribution of 17 blood group systems also was obtained. STUDY DESIGN AND METHODS: DNA samples from 200 Chinese donors were extracted and genotyped using the blood-MLPA assay. To confirm the MLPA results, a second independent genotyping assay (ID Core+) was conducted in 40 donors, and serological typing of 14 blood-group antigens was performed in 91 donors. In donors who had abnormal copy numbers of an allele (DI and GYPB) determined by MLPA, additional experiments were performed (polymerase chain reaction, sequencing, and flow cytometry analysis). RESULTS: The genotyping results obtained using the blood-MLPA and ID Core+ assays were consistent. Serological data were consistent with the genotyping results except for one donor who had a Lu(a-b-) phenotype. Of the 17 blood group systems, the distribution of the MNS, Duffy, Kidd, Diego, Yt, and Dombrock systems was polymorphic. The Mur and Sta antigens of the MNS system were distributed with a frequency of 9% (18 of 200) and 2% (4 of 200), respectively. One donor with chimerism and one who carried a novel DI*02(A845V) allele, which predicts the depression of Dib antigen expression, were identified. CONCLUSIONS: The blood-MLPA assay could easily identify the common blood-group alleles and correctly predicted phenotype in the Chinese population. The Mur and Sta antigens were distributed with high frequency in a Southern Chinese Han population.

Identification of a novel frequent RHCE*ce308T variant allele in Chinese D- individuals, resulting in a C+c- phenotype.

BACKGROUND: The RHCE allele is highly polymorphic; more than 60 variants have been described leading to diminished expression of C, c, E, and e antigens. Not much is known about the prevalence of RHCE variants in the Chinese population. Individuals carrying a variant are at risk to develop alloantibodies in response to mismatched pregnancy or transfusion. In this study, phenotyping and genotyping of the RHCE allele in Chinese donors revealed a new clinically relevant mutation. STUDY DESIGN AND METHODS: Blood samples from 200 D- and 200 D+ Chinese donors were analyzed by the RH multiplex ligation-dependent probe amplification (MLPA) assay and compared to serologically typed RhCE phenotypes, when available. All exons of the RHCE gene were sequenced in samples with aberrant genotyping results. The phenotype of the new variant RHCE allele was tested by transducing cultured human erythroblasts. RESULTS: Aberrant copy numbers for Exon 2 of the RHCE gene were discovered by MLPA in six D- donors (6/200), but not in D+ donors (0/200). Sequencing of the RHCE gene in these six donors identified a new variant RHCE*ce308C>T (p.103Pro>Leu) allele with an allele frequency of 0.015 within the D- individuals in this study. This variant was not detected in D+ individuals showing linkage with the D- haplotype. Serologically weak C expression and loss of c expression was demonstrated on donor red blood cells. In vitro transfection studies of the RHCE*ce308T variant in cDe/ce and CDe/CDe erythroblasts confirmed that the variant is associated with anti-C reactivity while abolishing c expression. CONCLUSION: Genotyping of individuals carrying this variant by standard RHCE genotyping might falsely predict a C- phenotype or a c+ phenotype. This new variant should be taken into account in RHCE genotyping assays designed for the Chinese population.

[Expression of PSF1 in colon cancer tissues and its effect on the proliferation of colon cancer cells].

OBJECTIVE: To detect the expression of PSF1 (partner of Sld five 1) in colon cancer specimens, and to explore the effect of RNA interference targeting PSF1 on the proliferation of colon cancer cells and its mechanism. METHODS: Expression level of PSF1 protein in colon cancer specimens was detected by Western blot in 40 patients with colon cancer from May 2004 to December 2006. The short hairpin RNA (shRNA) plasmid targeting PSF1 was transfected into LOVO, HT-29 and HCT116 cells with liposome, then the expression level of PSF1 protein was measured by Western blot, the effect of PSF1 shRNA plasmid transfection on cell proliferation by MTT assay, anchorage-independent growth by soft agar colomy-formation assay, and PSF2, PSF3 and SLD5 mRNA expression by quantitative reverse transcription polymerase chain reaction. RESULTS: The relative expression level of PSF1 protein in colon cancer tissues was 0.485±0.113, which was significantly higher than that in adjacent normal mucosa tissues (0.056±0.014, P<0.01). Western blot showed that the expression level of PSF1 protein was significantly decreased in colon cancer cells transfected with PSF1 shRNA plasmid. After PSF1 shRNA plasmid transfection, cell proliferation was significantly suppressed, the soft agar colony-forming rates of LOVO, HT-29 and HCT116 cells were significantly lower than those in control groups (P<0.05), meanwhile the expression levels of PSF2, PSF3 and SLD5 mRNA were significantly decreased (P<0.05). CONCLUSIONS: PSF1 is significantly up-regulated in colon cancer tissues compared with adjacent normal mucosa tissues. ShRNA plasmid targeting PSF1 can inhibit the expression of PSF1 gene, suppress the proliferation of colon cancer cells, suggesting that it may be a new therapeutic target for colon cancer.

[Expression and clinical significance of GINS complex in colorectal cancer].

OBJECTIVE: To investigate the expression and clinical significance of GINS complex in colorectal cancer (CRC). METHODS: The expression level of GINS complex including PSF1, PSF2, PSF3 and SLD5 in CRC specimens (n=76) were detected by real-time fluorescent quantitative polymerase chain reaction. The association of GINS complex with clinicopathological parameters and prognosis of CRC patients were analyzed. RESULTS: The relative expression level of PSF1, PSF2, PSF3, and SLD5 mRNA in CRC tissues was 0.001 853±0.000 651, 0.007 757±0.004 260, 0.000 967±0.000 481 and 0.003 248±0.001 721, which was significantly higher than that in normal colorectal mucosa tissues (0.000 352±0.000 169, 0.002 951±0.001 216, 0.000 472±0.000 271, and 0.001 675±0.001 156) (all P<0.01). PSF1 mRNA expression was associated with tumor size (P<0.01), and PSF2 mRNA expression with age (P<0.05) and lymph node metastasis (P<0.05). No correlations between PSF3 mRNA expression and clinicopathological parameters were observed. SLD5 mRNA expression was associated with lymph node metastasis (P<0.01). Patients with high expression of PSF1, PSF2 and SLD5 had worse 5-year overall survival rate (57.1%, 54.3%, and 54.3%) than those with low expression (77.1%, 80.0%, and 80.0%) (all P<0.05). Multivariable Cox regression analysis indicated that PSF1 mRNA expression (P<0.05) was an independent factor associated with prognosis of colorectal cancer. CONCLUSIONS: Overexpression of GINS complex in CRC is associated with clinicopathological characteristics and prognosis of colorectal cancer. PSF1 expression is prognostic for CRC patients.

Characterization of hepatitis virus B isolated from a multi-drug refractory patient.

Prolonged treatment of chronic hepatitis B (CHB) with nucleoside analogues (NAs) almost invariably engenders viral resistance, and sequential NAs monotherapy can promote multi-drug resistance. This study aimed to investigate the molecular characteristics and the mutation profile of multi-drug resistant hepatitis B virus (HBV). The complete genome of HBV isolated from a multi-drug refractory patient was amplified and cloned, and 22 clones were selected for sequencing. The homology of the full-length genome between clones ranged from 98.7% to 99.9%. A precore stop codon mutation of G1896A and basic core promoter (BCP) mutations A1762T/G1764A were detected in a majority of clones. A phylogenetic analysis showed that all clones were classified as subgenotype B2. Three mutations in the surface (S) antigen region, sC76Y, sP120T and sI195M, were detected in 100%, 100% and 77.3% of the clones, respectively. In the core (C) antigen region, a mutation at codon 135 (cP135Q) was detected in 100% of clones. Lamivudine (LAM)-resistant mutations, rtL180M and rtM204V/I were detected in 86.4% of clones. Adefovir (ADV) or entecavir (ETV)-resistant mutations were not detected. Several novel mutations, such as rtT128N, rtA222T, rtS256G, rtL271M, rtS332R, and rtN/T337D, were present in a majority of clones. Furthermore, six pairs of mutations in the overlapping reverse transcriptase (RT) gene and S gene were detected. In conclusion, the complex HBV mutation profile detected in the multi-drug refractory patient highlights the problems associated with the ongoing selection of mutations, including further compensatory mutations as well as potential cross-resistance mutations.