[Molecular study of an individual with Bel subtype due to a novel c.620T>C variant].

OBJECTIVE: To explore the molecular basis for an individual with Bel subtype of the ABO blood type due to a novel c.620T>C variant gene, and assess its impact on the structure of GTB transferase. METHODS: An individual who had visited the First Affiliated Hospital of Zhengzhou University on February 11, 2023 was selected as the study subject. ABO phenotyping was initially conducted with serological methods, which was followed by direct sequencing of 7 exons of the ABO gene. Subsequently, single-strand sequencing was carried out by using allele-specific primers, and the variant in the B transferase was homology-modeled using the Modeller software. The impact of the variant on the transferase's spatial structure was analyzed with the PyMOL software. RESULTS: The serological phenotype of the patient was identified as the Bel subtype. Direct sequencing revealed that she has harbored a novel c.620T>C variant, resulting in a p.Leu207Pro substitution in the polypeptide chain. Combined with single-strand sequencing, her genotype was ultimately determined as ABO*BELnew/ABO*O.01.02. Three-dimensional protein structure modeling showed that, compared with the wild type, the distance of one hydrogen bond between Proline and Glycine at position 272 has increased, along with disappearance of another hydrogen bond. CONCLUSION: The novel c.620T>C (p.Leu207Pro) variant of B allele may affect the structural stability of the glycosyltransferase. The weakened enzyme activity in turn may lead to reduced B antigen expression, manifesting as the Bel subtype by serological analysis.

[The Results of ABO/RhD Blood Group and Comparative Analysis Detected by Two Methods in Infants Younger than 6 Months].

OBJECTIVE: To detect the ABO / RhD blood type of infants younger than 6 months in different gestational age and month old with automatic microcolumn glass sphere and tube method, and compare the result of the two methods. METHODS: The data of 896 samples of infants younger than 6 months from January 2018 to February 2019 was collected. The two methods were used to detect ABO/RhD blood type in all samples and compare the detection rate of ABO/RhD antigen and ABO reverse typing and agglutination intensity of the two methods. RESULTS: Three hundred and eight cases of type A (34.4%), 281 cases of type B (31.4%), 210 cases of type O (23.4%), 97 cases of type AB (10.8%), and 896 positive cases of RhD blood type were detected out by two methods. There were no significant differences of ABO/RhD antigen agglutination intensity between two methods (P > 0.05). Except for type AB, the detection rate of ABO reverse typing in infants with type B was significantly higher than that with type A and type O (P < 0.05). The agglutination intensity of type A reverse cell was higher than type B reverse cell (P < 0.05). The fully automatic microcolumn glass sphere method exhibited higher detection rate of ABO reverse typing in the samples of type A and type O group and agglutination intensity of ABO reverse typing in all types as compared with the tube method (P < 0.05). The detection rate and agglutination intensity of ABO reverse typing in term group were significantly higher than those in preterm group (P < 0.05). The fully automatic microcolumn glass sphere method exhibited higher detection rate of ABO reverse typing and agglutination intensity compared with the tube method between two groups (P < 0.05). The detection rate and agglutination intensity of ABO reverse typing in group IV (4-6 months old) were significantly higher than those in groups I, II and III (young than 3 months old) (P < 0.05). The fully automatic microcolumn glass sphere method exhibited higher detection rate of ABO reverse typing in I, II, III groups and agglutination intensity of ABO reverse typing in the 4 groups compared with the tube method (P < 0.05). CONCLUSION: ABO / RhD blood group antigen can be accurated detected in majority of infants, but the detection rate of ABO antibody is related to gestational age and month age of infants. The detection rate and agglutination intensity of the fully automatic microcolumn glass sphere method in ABO reverse typing are higher than those of the tube method, especially for premature infants and children within 3 months old.

[Genotyping for Auxiliary Identification of Anti-C Alloantibody with Anti-f Autoantibody Mimicking Alloantibody].

OBJECTIVE: To identify the difficult antibody specificity of 1 PNH patient with blood transfusion history by genotyping. METHODS: RH typing of this patient was performed using gel card method, the antibody specificity was identified by panel cells, the RH-unrelated phenotype were excluded by genotyping method in difficult condition of serologic identification, furthmore different RH phenotype cells were used for adsorption-elution so as to re-examine the reactivity of antibodies in this patient's serum, and finally different RH phenotype cells were combined to exclude other unrelated antibodies. RESULTS: The RH phenotype presented as double population for C antigen, and positive agglutination for the other antigens. The results of RHD zygote, together with RHD and RHCE sequencing showed that the RHD genotype was homozygous RHD/RHD, and the c.122A＞G mutation did not found in RHCE gene, thus CW antigen was excluded; 48G lies in the 1st exon, the 5th exon showed nt676 G/C heterozygosity, the 2nd-4th, 6th-10th exons did not show mutations, however a new mutation was found in the 4th intron IVS4+29A＞C. It can be judged that the RH genotype was Dce/DcE, and its phenotype should be ccDEe. The anti-C alloantibody and rare anti-f autoantibody mimicking alloantibody in serum was determined by different RH phenotypic cell absorption-elution. Finally, cross-matched AB ccDEE blood units were selected for the patient and there was no adverse reactions of blood transfusion occurred. CONCLUSION: Genotyping can be auxiliarily applied to the identification of difficult antibodies in serum of a patient, thereby reducing the risk of blood transfusion.

[Characterization of a novel allele of Aw33 subtype of the ABO blood group].

OBJECTIVE: To explore the molecular basis for an A subtype of the ABO blood group. METHODS: The forward and reverse typing of the ABO blood group were identified by gel card and test tube methods. The ABO gene of the patient was detected by PCR-sequence specific primer (PCR-SSP). Exons 1 to 7 of the ABO gene was amplified by PCR and sequenced. The ABO gene was also subjected to subclone sequencing for haplotype analysis. RESULTS: The patient's red cells showed weak agglutination with anti-A but non-agglutination with anti-B. The patient's serum showed 1+ agglutination with A cells and 4+ agglutination with B cells. Based on above serological characteristics, the patient was defined as Aw subtype of the ABO blood group. Sequencing analysis showed that the patient was heterozygous for c.106G>T, c.188G>A, c.189C>T, c.220C>T, c.297A>G, c.467C>T, c.543G>C, c.646T>A, c.681G>A, c.771C>T, c.829G>A, in addition with a c.261G deletion. Combined with the result of subclone sequencing, the ABO genotype of the patient was determined as ABO*AW.33. new/O.01.02, which harbored c.467C>T and c.543G>C variants compared with ABO*A1.01 and c.543G>C variant compared with ABO*A1.02. The novel allele has been submitted to GenBank with an accession number of MK302122. CONCLUSION: A novel allele of Aw33 subtype has been identified with its GTA transferase gene harboring c.467C>T and c.543G>C variants compared with A1.01.

Timely diagnosis and treatment of neonatal alloimmune thrombocytopenia caused by anti HPA-3a antibody: A case report.

RATIONALE: Neonatal alloimmune thrombocytopenia (NAIT) caused by anti HPA-3a antibody is rare, and the clinical features of the syndrome are not specific. PATIENT CONCERNS: A male infant was noted to be irritable and physical examination revealed the presence of petechiae and bruising on the right arm and thigh after born. DIAGNOSES: Platelet antibodies were investigated using the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, platelet genotyping (HPA 1-17) was performed by polymerase chain reaction technique with sequence-specific primers (PCR-SSP). The HPA genotype of the newborn was HPA-3a/b, while that of his mother and his father were HPA-3b/b and HPA-3a/a, respectively. The sera of newborn contained antibody against the platelet of newborn's father. The HPA antibody of the newborn was identified as anti HPA-3a. The newborn was confirmed as a patient of NAIT caused by anti HPA-3a antibody. INTERVENTIONS: A single dose of intravenous immunoglobulin (IVIG) 1 g/kg was administered from day 3 to day 7. OUTCOMES: At follow-up 3 months after discharge from the hospital, the baby was developing normally and had a normal platelet count (361 × 109/L). LESSONS: NAIT caused by anti HPA-3a antibody is rare, and we believe this study can provide insights for diagnosing prospective cases. Prognosis of NAIT caused by HPA3a seems to be favorable if diagnosed and treated in a timely manner.

LncHOXA10 drives liver TICs self-renewal and tumorigenesis via HOXA10 transcription activation.

BACKGROUND: Liver cancer is one of the most deadly cancers in the world. There are various cells in liver tumor bulk, including liver tumor initiating cells (TICs), which account for liver tumorigenesis, drug resistance, relapse and metastasis. The homeobox (HOX) transcription factors play critical roles in many physiological and pathological processes, while, their roles in liver TICs and liver tumorigenesis remain unknown. METHODS: An unbiased screening was performed using online-available datasets. Liver TICs were sorted by FACS using surface markers CD133, CD13 and EPCAM, or enriched by oncosphere formation assay. TIC self-renewal was examined by oncosphere formation and tumor initiation assay. Loss of function and gain of function assays were performed to examine the role of lncRNA. RNA pulldown, RNA immunoprecipitation, ChIP, Western blot and double FISH were used to explore the molecular mechanism of lncRNA. RESULTS: Here, we examined the expression pattern of HOX transcription factors, and found HOXA10 was overexpressed in liver cancer samples. Moreover, a divergent lncRNA of HOXA10 (termed lncHOXA10 hereafter) was also highly expressed in liver cancer and liver TICs. LncHOXA10 drove liver TIC self-renewal and liver tumorigenesis through HOXA10-dependent manner. LncHOXA10 interacted with SNF2L and recruited NURF chromatin remodeling complex to HOXA10 promoter, and thus initiated the transcription of HOXA10. Through HOXA10 transcriptional regulation, lncHOXA10 activated HOXA10 in liver TICs. LncHOXA10-HOXA10 signaling can be targeted to eliminate liver TICs. Altogether, lncHOXA10 drove HOXA10 expression and thus promoted liver TIC self-renewal. CONCLUSION: HOXA10 was the most highly expressed HOX transcription factor in liver cancer and liver TICs. LncHOXA10 drove the transcriptional activation of HOXA10. This work revealed the important role of HOX transcription factor in liver TIC self-renewal and added a new layer for liver TIC regulation.

LncRNA TUG1 interacting with miR-144 contributes to proliferation, migration and tumorigenesis through activating the JAK2/STAT3 pathway in hepatocellular carcinoma.

Recently, it is reported that taurine upregulated gene 1 (TUG1) participates in the tumor progression by acting as a competing endogenous RNA (ceRNA) of miRNAs. Nonetheless, whether TUG1 could serve as a ceRNA of miR-144 in hepatocellular carcinoma (HCC) progression remains undefined. Here, our results indicated that there was a marked rise in TUG1 expression in HCC tissues and cells, and downregulation of TUG1 hindered proliferation and migration of HCC cells. Additionally, TUG1 was validated to act as a molecular sponge of miR-144. Furthermore, we found that TUG1 interacting with miR-144 contributed to proliferation and migration of HCC cells via activating the JAK2/STAT3 pathway in vitro. Moreover, TUG1 knockdown inhibited HCC tumor growth in vivo through upregulating miR-144 via inactivation of the JAK2/STAT3 pathway. In conclusion, TUG1 interacting with miR-144 contributed to proliferation, migration and tumorigenesis through activation of the JAK2/STAT3 pathway in HCC.

MicroRNA-30a Mediates Cell Migration and Invasion by Targeting Metadherin in Colorectal Cancer.

MicroRNAs play critical roles in the occurrence and progression in various cancers including colorectal cancer. Here, we found that microRNA-30a expression was significantly downregulated in colorectal cancer tissues compared to adjacent noncancerous tissues, and the suppression levels of microRNA-30a were significantly associated with tumor differentiation and lymph node metastasis. We also discovered that the expression level of microRNA-30a was inversely proportional to the invasive potential of several colorectal cancer cell lines. Moreover, overexpression of microRNA-30a in colorectal cancer cells inhibited activity of cell migration and invasion. Luciferase reporter assay confirmed metadherin could be a direct target of microRNA-30a, as the overexpression of microRNA-30a decreased metadherin expression at both the protein and messenger RNA levels. Furthermore, the knockdown of metadherin expression in SW620 significantly decreased cell metastasis and invasion. The upregulation of metadherin at the protein level negatively correlated with the expression of microRNA-30a in colorectal cancer tissues, and this upregulation could partially attenuate the effect induced by microRNA-30a. These findings indicate that microRNA-30a may act as a tumor suppressor in colorectal cancer and that microRNA-30a represses cell migration and invasion by decreasing metadherin, highlighting the therapeutic potential of microRNA-30a and metadherin in colorectal cancer treatment.

[Comparison of Weak ABO Antigen and Normal ABO Antigen in Patients with Acute Leukemia].

OBJECTIVE: To compare the differences between weak ABO antigen patients and normal ABO antigen patients with acute leukemia, and to explore the clinical significance of weak ABO antigen in acute leukemia. METHODS: The ABO blood group was detected in 110 newly diagnosed acute leukemia patients(including 68 cases of AML and 42 cases of ALL) and 68 normal controls. Then the leukemia subtype, age, sex, laboratory test, risk status of leukemia patients, and DNA methylation of ABO promoter were compared between patients with weak and normal ABO antigen. RESULTS: The weak ABO antigen was found in patients with newly diagnosed acute leukemia, and was not found in ALL patients or normal group. No statistical differences were found in the distribution of ABO blood group, age, hepatosplenomegaly, lymphadenovarix, plt, precursor cell clusters derived from bone marrow, immunopheno-typing, LDH level, and risk status between AL patients of weak and normal ABO antigen groups (P>0.05). Compared with patients in normal ABO antigen group, the pateins in weak ABO antigen group had higher percentage of male(77.8% vs 30%), lower WBC(32.26×109/L vs 82.69×109/L) and Hb level(64.00 g/L vs 85.94 g/L) and higher DNA methylation level (18.91% vs 10.76%) (P<0.05). CONCLUSION: The cases of weak ABO antigen frequently appear in the male AML patients, the DNA methylation level of ABO gene promoter in patients with weak ABO antigen is significantly higher than that in patients with normal ABO antigen.

Absolute Eosinophil Count was Significantly Associated with Serum (1,3)-ß-D-Glucan in Patients with Invasive Fungal Infections.

BACKGROUND: Measurement of serum (1,3)-ß-D-glucan has emerged as an important diagnostic strategy for invasive fungal infections (IFI). Previous studies suggested that the eosinophil count would be related with the value of serum (1,3)-ß-D-glucan. METHODS: 572 blood samples obtained from 126 patients at the First Affiliated Hospital of Zhengzhou University were analyzed. The unsuitable samples were excluded according to the patient selection. RESULTS: In our study, we found that in the 572 cumulative results, the absolute eosinophil count was significantly associated with the value of (1,3)-ß-D-glucan (r = 0.57; 95% confidence interval (CI) 0.51 - 0.63) but not with total leukocyte, neutrophils, lymphocytes, monocytes, and basophils. Then, we found that there were 126 patients with invasive fungal infections, 109 patients with candidiasis and 17 patients with aspergillosis. In the 109 patients who had candidiasis, absolute eosinophil count was significantly associated with the value of (1,3)-ß-D-glucan (r = 0.59; 95% CI 0.53 - 0.65). Similarly, in the 17 patients who had aspergillosis, the absolute eosinophil count was significantly associated with the value of (1,3)-ß-D-glucan (r = 0.65; 95% CI 0.51 - 0.75). Meanwhile, similar findings in different types of Candida species or Aspergillus species in patients were found except in Candida glabrata infection. CONCLUSIONS: As a routine and broadly used test, serial absolute eosinophil count is related with IFI, and it could be used to evaluate the progress of IFI.

[A single E726Q mutation in the membrane proximal α-helix of integrin ß3 subunit induces membrane blebbing by disrupting the membrane-actin cortex interaction].

The membrane proximal α helix of integrin ß subunit cytoplasmic tails plays an important functional role by interacting with various intracellular proteins, namely talin, α-actinin or skelemin. This study was designed to investigate the functional role of 5 highly conserved charged amino acids (R(724), K(725), E(726), E(731), E(733)) within this α helix by site-directed mutagenesis. The result showed that CHO cells expressing the αIIbß3E726Q mutant had the most prominent phenotype and characterized by defective cell spreading on immobilized fibrinogen. In addition, this E726Q mutation induced membrane blebbing in cells adherent on fibrinogen, and this blebbing could be inhibited by the myosin light chain ATPase inhibitor blebbistatin. It is concluded that the membrane proximal α-helix of integrin ß3 subunit is important in linking the phospholipid membrane to the submembraneous actin cortex.