COX7B Is a New Prognostic Biomarker and Correlates with Tumor Immunity in Esophageal Carcinoma.

Esophageal carcinoma (ESCA) refers to the most common type of malignant tumor, which reveals that it occurs often all over the world. ESCA is also correlated with an advanced stage and low survival rates. Thus, the development of new prognostic biomarkers is an absolute necessity. In this study, the aim was to investigate the potential of COX7B as a brand-new predictive biomarker for ESCA patients. COX7B expression in pancancer was examined using TIMER2. The statistical significance of the predictive value of COX7B expression was explored. The relationship between COX7B expression and tumor-infiltrating immune cells in ESCA was analyzed by using ssGSEA. In this study, the result indicated that several types of cancers had an abnormally high amount of COX7B. COX7B expression in samples from patients with ESCA was considerably higher than in nontumor tissues. A more advanced clinical stage may be anticipated from higher COX7B expression. According to the findings of Kaplan-Meier survival curves, patients with low COX7B levels had a more favorable prognosis than those with high COX7B levels. The result of multivariate analysis suggested that COX7B expression was a standalone prognostic factor for the overall survival of ESCA patients. A prognostic nomogram including gender, clinical stage, and COX7B expression was constructed, and TCGA-based calibration plots indicated its excellent predictive performance. An analysis of immune infiltration revealed that COX7B expression has a negative correlation with TFH, Tcm, NK cells, and mast cells. COX7B may serve as an immunotherapy target and as a biomarker for ESCA diagnosis and prognosis.

Identification of a compound heterozygote in LYST gene: a case report on Chediak-Higashi syndrome.

BACKGROUND: Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease caused by loss of function of the lysosomal trafficking regulator protein. The causative gene LYST/CHS1 was cloned and identified in 1996, which showed significant homology to other species such as bovine and mouse. To date, 74 pathogenic or likely pathogenic mutations had been reported. CASE PRESENTATION: Here we describe a compound heterozygote in LYST gene, which was identified in a 4-year-old female patient. The patient showed skin hypopigmentation, sensitivity to light, mild splenomegaly and reduction of platelets in clinical examination. Giant intracytoplasmic inclusions were observed in the bone marrow examination, suggesting the diagnosis of CHS. Amplicon sequencing was performed to detect pathogenic mutation in LYST gene. The result was confirmed by two-generation pedigree analysis base on sanger sequencing. CONCLUSION: A compound heterozygote in LYST gene, consisting of a missense mutation c.5719A > G and an intron mutation c.4863-4G > A, was identified from the patient by using amplicon sequencing. The missense mutation is reported for the first time. Two-generation pedigree analysis showed these two mutations were inherited from the patient's parents, respectively. Our result demonstrated that amplicon sequencing has great potential for accelerating and improving the diagnosis of rare genetic diseases.

Genomic and biological characterization of a pandemic norovirus variant GII.4 Sydney 2012.

Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.

[Phenotypic and genetic analysis of a boy with partial trisomy of 22q].

OBJECTIVE: To delineate the nature and origin of chromosomal aberration in a boy with mental retardation and multiple congenital deformities. METHODS: Chromosomal karyotypes of the proband and his parents were determined by routine G-banding analysis. Genomic DNA was also analyzed with single nucleotide polymorphism array (SNP array). RESULTS: The karyotype of the proband was 46,X,add(Y)(q11.23). No karyotypic abnormality was detected in either parent. SNP array has identified a de novo 21.6 Mb duplication at 22q12qter in the proband. CONCLUSION: The de novo 22q12qter duplication probably underlies the abnormalities in the proband.

[Clinical and genetic analysis of a case with infantile Parkinsonism with motor delay due to tyrosine hydroxylase deficiency].

OBJECTIVE: To explore the clinical characteristics and genetic variants in a child with tyrosine hydroxylase-deficient infantile Parkinsonism with motor delay. METHODS: Clinical feature of the patient was summarized. Genomic DNA was extracted from peripheral blood samples taken from the child and her family members. All exons of GCH1, TH and SPR genes were subjected to targeted capture and next-generation sequencing. Suspected variants were verified by Sanger sequencing. RESULTS: The child could not sit alone at 7 month and 11 days. Physical examination suggested motor retardation and hypotonia, limb stiffness, head nodding, slight torticollis, and language and intellectual developmental delays. She developed involuntary shaking of limbs at 3 month old, which lasted approximately 10 seconds and aggregated with excitement and before sleeping. Cranial MRI revealed widening of subarachnoid space on the temporomandibular and particularly temporal sides. Genetic testing revealed that she has carried a nonsense c.457C>T (p.R153X) variant, which was known to be pathogenic, and a novel missense c.720C>G (p.I240M) variant of the TH gene. The two variants were derived from her father and mother, respectively. CONCLUSION: The child was diagnosed as tyrosine hydroxylase-deficient infantile Parkinsonism with motor delay due to compound heterozygous variants of the TH gene. Above finding has enriched the spectrum of TH gene variants.

[Phenotypic and genetic analysis of a boy with a 10p15.3 deletion and partial trisomy 18p syndrome].

OBJECTIVE: To delineate the nature and origin of chromosomal aberration in a girl with mental retardation. METHODS: Genomic DNA was analyzed by using single nucleotide polymorphism-based array (SNP array). The proband and her parents were subjected to routine G-banded chromosomal karyotyping analysis. RESULTS: SNP array has identified a 1.2 Mb microdeletion at 10p15.3 and a duplication at 18p11.21-pter in the proband. The patient was also found to harbor a structural aberration involving 10p. The karyotype of her father was 46,XY,t(10;18)(p15;p11.2), while her mother was found to be normal. CONCLUSION: The structural aberration of 10p carried by the patient has derived from her father whom has carried a balanced translocation of t(10;18). Her karyotype was finally determined as 46,XX,der(10)t(10;18)(p15;p11.2)pat. The abnormal phenotype of the patient can probably be attributed to the presence of 10p15.3 microdeletion and 18p11.21-pter duplication.

[Clinical and genetic study of a child with delayed language development carrying ring 22 and a 22q13 microdeletion].

OBJECTIVE: To explore the genetic basis for a child featuring delayed language development. METHODS: The patient was subjected to conventional G-banding chromosomal karyotyping and single nucleotide polymorphism microarray (SNP array) analysis. RESULTS: The karyotype of the child was 46, XY, r(22)(p11.2q13). SNP array analysis has identified a hemizygous 1.67 Mb deletion at 22q13 (arr [Hg19]22q13.33 (49 531 302-51 197 766)×1). CONCLUSION: The child has carried a ring 22 in addition with a 22q13 microdeletion. The results may provide clues for her condition and genetic counseling for the family.

H19 suppresses the growth of hepatoblastoma cells by promoting their apoptosis via the signaling pathways of miR-675/FADD and miR-138/PTK2.

BACKGROUND: The objective of this study was to clarify the molecular pathways involved in hepatitis B virus (HBV)-induced hepatoblastoma. METHOD: The expression of factors in different signaling pathways (H19, miR-675, miR-138, protein tyrosine kinase 2 [PTK2], fas-associated death domain [FADD], hypoxia-inducible factor 1-alpha [HIFIA], focal adhesion kinase [FAK], caspase-8, and caspase-3) was compared between HBV (+) and HBV (-) groups using quantitative real-time polymerase chain reaction and Western blot analysis. Subsequently, immunohistochemistry (IHC) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays were used to verify the expression of above proteins in HBV (+) and HBV (-) groups. Computational analysis was conducted to predict the target genes of miR-675 and miR-138, whose regulatory relationships were then clarified using luciferase assays and cell transfection studies. RESULT: The expression of H19, miR-675, PTK2, HIFIA, and FAK was increased in the HBV (+) group, while the expression of miR-138, FADD, caspase-8, and caspase-3 was decreased in the HBV (+) group. FADD and PTK2 were identified as target genes of miR-675 and miR-138, respectively. In addition, miR-675 was upregulated while miR-138 was downregulated by X protein (HBx). CONCLUSION: In summary, the results of this study revealed the molecular pathways involved in HBV-induced hepatoblastoma. In the presence of HBV, HBX upregulated the expression of H19 through HIFIA. Consecutively, overexpressed H19 upregulated the expression of PTK2 via targeting miR-138 and downregulated the expression of FADD via targeting miR-675. Finally, increased expression of PTK2 and reduced expression of FADD both led to the inhibition of cell apoptosis, thus promoting the tumorigenesis of hepatoblastoma.

[Clinical and genetic diagnosis of a pedigree affected with autosomal recessive Alport syndrome].

OBJECTIVE: To explore the genetic basis of a child with chronic kidney disease featuring renal shrinkage and creatinine increase. METHODS: Peripheral venous blood samples were taken from the child, his brother and two parents and subjected to whole exome sequencing. Suspected mutations were verified by Sanger sequencing. Bioinformatic analysis was carried out to predict the influence of mutations on the structure and function of the protein product. RESULTS: High-throughput and Sanger sequencing revealed that the child has carried compound heterozygous mutations of the COL4A4 gene, namely c.4550T>G in exon 47 (inherited from his mother) and c.199C>T in exon 5 (inherited from his father). Neither mutation was reported previously. Bioinformatic analysis showed that both mutations have located in highly conserved regions. The same mutations were not found in his brother. CONCLUSION: The compound heterozygous c.4550T>G and c.199C>T mutations probably underlie the disease in this child. The findings have enriched the mutation spectrum of the COL4A4 gene.

[Morphology and pathogenesis of 47, XYY/47, XY, +mar identified in patients with super male syndrome].

OBJECTIVE: To explore the source and morphology of supernumerary markers from patients with 47,XYY/47,XY, +mar and supermale syndrome. METHODS: Conventional GTG banded karyotyping and dual-color fluorescence in situ hybridization (FISH) were performed on 21 such patients. RESULTS: Among these cases, 18 had their small supernumerary marker derived from the Y chromosome. Three were derived from autosomal chromosomes. Those derived from Y chromosome were small fragments with centromeres, while those derived from autosomes were in the ring form. CONCLUSION: In children with supermale syndrome and 47,XYY/47,XY,+ mar, the supernumerary marker chromosomes primarily derive from sex chromosomes. These small chromosomes mainly have the forms of small segments with centromeres or rings. For such children, molecular cytogenetic analysis can facilitate genetic counseling and prenatal diagnosis.

[Identification of a novel splicing mutation in COL1A1 gene in a Chinese family affected with typeâ osteogenesis imperfecta].

OBJECTIVE: To investigate the genetic cause for a large family affected with typeⅠosteogenesis imperfecta. METHODS: Genomic DNA was extracted from peripheral venous blood samples. The entire coding region and intron-exon boundaries of the COL1A1 gene were subjected to PCR amplification and direct sequencing. Total RNA was also extracted from immortalized B cell lines from the patients, with the first strand of cDNA synthesized with an oligo(dT)18 primer. The PCR products were directly sequenced using the TA cloned plasmid. RESULTS: A c.3208G>A mutation has been identified in the COL1A1 gene, which can alter the splicing pattern of mRNA. CONCLUSION: A novel splicing mutation c.3208G>A of the COL1A1 gene probably underlies the disease.

MICAL1 promotes the proliferation in acute myeloid leukemia and is associated with clinical prognosis and immune infiltration.

Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies that has a poor prognosis and a high rate of relapse. Dysregulated metabolism plays an important role in AML progression. This study aimed to conduct a comprehensive analysis of MRGs using TCGA and GEO datasets and further explore the potential function of critical MRGs in AML progression. In this study, we identified 17 survival-related differentially expressed MRGs in AML using TCGA and GEO datasets. The 150 AML samples were divided into three molecular subtypes using 17 MRGs, and we found that three molecular subtypes exhibited a different association with ferroptosis, cuproptosis and m6A related genes. Moreover, a prognostic signature that comprised nine MRGs and had good predictive capacity was established by LASSO-Cox stepwise regression analysis. Among the 17 MRGs, our attention focused on MICAL1 which was highly expressed in many types of tumors, including AML and its overexpression was also confirmed in several AML cell lines. We also found that the expression of MICAL1 was associated with several immune cells. Moreover, functional experiments revealed that knockdown of MICAL1 distinctly suppressed the proliferation of AML cells. Overall, this study not only contributes to a deeper understanding of the molecular mechanisms underlying AML but also provides potential targets and prognostic markers for AML treatment. These findings offer robust support for further research into therapeutic strategies and mechanisms related to AML, with the potential to improve the prognosis and quality of life for AML patients. Nevertheless, further research is needed to validate these findings and explore more in-depth molecular mechanisms.

Luteolin enhances drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in paclitaxel­resistant esophageal squamous cell carcinoma.

Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem­like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti­tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)­resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit­8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT­related proteins and stem cell markers after sphere formation. Parental cells and drug­resistant cells were screened by high­throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX­resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug­resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX­resistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX­resistant ESCC and could be a promising agent for the treatment of PTX­resistant ESCC cancers.

Comprehensive Molecular Analyses of Notch Pathway-Related Genes to Predict Prognosis and Immunotherapy Response in Patients with Gastric Cancer.

Gastric cancer (GC) is a highly molecular heterogeneous tumor with unfavorable outcomes. The Notch signaling pathway is an important regulator of immune cell differentiation and has been associated with autoimmune disorders, the development of tumors, and immunomodulation caused by tumors. In this study, by developing a gene signature based on genes relevant to the Notch pathway, we could improve our ability to predict the outcome of patients with GC. From the TCGA database, RNA sequencing data of GC tumors and associated normal tissues were obtained. Microarray data were collected from GEO datasets. The Molecular Signature Database (MSigDB) was accessed in order to retrieve sets of human Notch pathway-related genes (NPRGs). The LASSO analysis performed on the TCGA cohort was used to generate a multigene signature based on prognostic NPRGs. In order to validate the gene signature, the GEO cohort was utilized. Using the CIBERSORT method, we were able to determine the amounts of immune cell infiltration in the GC. In this study, a total of 21 differentially expressed NPRGs were obtained between GC specimens and nontumor specimens. The construction of a prognostic prediction model for patients with GC involved the identification and selection of three different NPRGs. According to the appropriate cutoff value, the patients with GC were divided into two groups: those with a low risk and those with a high risk. The time-dependent ROC curves demonstrated that the new model had satisfactory performance when it came to prognostic prediction. Multivariate assays confirmed that the risk score was an independent marker that may be used to predict the outcome of GC. In addition, the generated nomogram demonstrated a high level of predictive usefulness. Moreover, the scores of immunological infiltration of the majority of immune cells were distinctly different between the two groups, and the low-risk group responded to immunotherapy in a significantly greater degree. According to the results of a functional enrichment study of candidate genes, there are multiple pathways and processes associated with cancer. Taken together, a new gene model associated with the Notch pathway may be utilized for the purpose of predicting the prognosis of GC. One potential method of treatment for GC is to focus on NPRGs.

Characteristics, polarization and targeted therapy of mononuclear macrophages in rheumatoid arthritis.

Macrophages are the core of the pathophysiology of rheumatoid arthritis (RA). They participate in specific and non-specific immunological responses, have phagocytosis, chemotaxis and immune regulatory functions, and are involved in the onset and progression of RA. In recent years, research on the pathophysiology of RA has focused on the polarization and functions of classically activated M1 and selectively activated M2 macrophage subtypes. M1 macrophages release different proinflammatory cytokines, thus driving the chronic proinflammatory, tissue destruction and pain response in RA. M2 macrophages play an anti-inflammatory role. Because of the important role of monocyte-macrophage in RA, drug research targeting monocyte-macrophage can bring us more hope for treatment of RA. This study reviewed the characteristics, plasticity, molecular activation mechanism and relationship of RA with mononuclear macrophages, as well as the transformative potential of macrophages in developing new therapeutic drugs for clinical practice.

[Expression Level and Correlation of miR-211, miR-155, C-myc in Acute T Lymphocytic Leukemia].

OBJECTIVE: To investigate the expression and correlation of miR-211, miR-155, and C-myc in acute T lymphocytic leukemia (T-ALL), aiming to provide evidence for the diagnosis and treatment. METHODS: A total of 96 T-ALL patients who were diagnosed and treated in People's Hospital of Zhengzhou from June 2014 to June 2017 were selected, and 69 healthy volunteers who had a physical examination were selected as control group in the same period. Real-time fluorescent quantitative PCR (RT-PCR) was used to determine the expression levels of miR-211, miR-155, and C-myc in peripheral blood mononuclear cells in each group. Kaplan-Meier was used to analyze the survival of T-ALL patients and correlation of miR-211, miR-155, and C-myc with prognosis. Pearson correlation analysis was used to evaluate the correlation of miR-211, miR-155, and C-myc with disease risk. RESULTS: The expression levels of miR-211 mRNA, miR-155 mRNA, and C-myc mRNA in T-ALL group were higher than those in the control group (P<0.01), those in non-remission group were higher than those in remission group (P<0.01), and those in high-risk group were also higher than those in low-risk group and intermediate-risk group (P<0.01). The survival time of T-ALL patients with low miR-211 expression was longer than that with high miR-211 expression (P<0.01), that with low miR-155 expression was longer than that with high miR-155 expression (P<0.01), and that with low C-myc expression was also longer than that with high C-myc expression (P<0.01). The high expression of miR-211, miR-155, and C-myc was linearly positively correlated with high risk of disease (r=0.749, 0.781, 0.804). CONCLUSION: The expressions of miR-211, miR-155, and C-myc are up-regulated in T-ALL patients, closely related to prognosis, and linearly positively correlated with disease risk.

[Effect of Curcumin on the Proliferation, Apoptosis, and Cell Cycle of Human Acute Myeloid Leukemia Cell Line K562].

OBJECTIVE: To investigate the effects of curcumin on the proliferation, apoptosis, and cell cycle of human acute myeloid leukemia cell line K562. METHODS: MTT method was used to detect the proliferation inhibition of logarithmic growth phase human acute myeloid leukemia K562 cells, flow cytometry was used to detect the cell cycle, Annexin V-FITC was used to detect the apoptosis rate, and real-time fluorescent quantitative PCR and Western blot were used to detect the expression of Bax, BCL-2 and caspase-3 mRNA and protein, respectively. RESULTS: The inhibition rate of cell proliferation in curcumin 10, 20, and 40 µmol/L group for 24 h and 48 h were higher than that in the control group (curcumin 0 µmol/L), and the cell proliferation inhibition rate was concentration-time dependent (r=0.879, r=0.914). The proportion of G0/G1 cells and apoptosis rate of K562 cells in the curcumin 10, 20, and 40 µmol/L group were higher than those in the control group, and showed drug concentration dependent (r=0.856, r=0.782). The expression of Bax and Caspase-3 mRNA in the curcumin 10, 20, and 40 µmol/L group was higher, while BCL-2 mRNA was lower than those in the control group, and showed drug concentration dependent (r=0.861, r=0.748, r=-0.817). The gray value of Bax protein expression in the curcumin 10, 20, and 40 µmol/L group was higher than that in the control group, while the gray value of BCL-2 and Caspase-3 protein expression was lower than that in the control group, and showed drug concentration dependent (r=0.764, r=-0.723, r=-0.831). CONCLUSION: Curcumin can inhibit the proliferation of human acute myeloid leukemia cell line K562 cells, block the cell cycle at G0/G1 phase, promote cell apoptosis, and induce apoptosis by regulating Bax, BCL-2, and Caspase-3.

Overexpression of Foxc1 ameliorates sepsis­associated encephalopathy by inhibiting microglial migration and neuroinflammation through the IκBα/NF­κB pathway.

Sepsis­associated encephalopathy (SAE) is a common and severe complication of sepsis. The cognitive dysfunction that ensues during SAE has been reported to be caused by impairments of the hippocampus. Microglia serves a key role in neuroinflammation during SAE through migration. Forkhead box C1 (Foxc1) is a member of the forkhead transcription factor family that has been found to regulate in cell migration. However, the role of Foxc1 in neuroinflammation during SAE remains unknown. In the present study, the mechanistic role of Foxc1 on microglial migration, neuroinflammation and neuronal apoptosis during the occurrence of cognitive dysfunction in SAE was investigated. A microglia­mediated inflammation model was induced by LPS in BV­2 microglial cells in vitro, whilst a SAE­related cognitive impairment model was established in mice using cecal ligation and perforation (CLP) surgery. Cognitive function in mice was evaluated using the Morris Water Maze (MWM) trial. Lipopolysaccharide (LPS) treatment was found to trigger BV­2 cell migration, inflammation and neuronal apoptosis. In addition, CLP surgery induced cognitive injury, which was indicated by longer latencies and shorter dwell times in the goal quadrant compared with those in the Sham group in the MWM trial. LPS treatment or CLP induction decreased the expression of Foxc1 and inhibitor of NF­κB (IκΒα) whilst increasing that of p65, IL­1ß and TNF­α. After Foxc1 was overexpressed, the cognitive dysfunction of mice that underwent CLP surgery was improved, with the expression of IκBα also increased, microglial cell migration, the expression of p65, IL­1ß and TNF­α and neuronal apoptosis were all decreased in vivo and in vitro, which were in turn reversed by the inhibition of IκBα in vitro. Overall, these results suggest that the overexpression of Foxc1 inhibited microglial migration whilst suppressing the inflammatory response and neuronal apoptosis by regulating the IκBα/NF­κB pathway, thereby improving cognitive dysfunction during SAE.

Next-generation sequencing and targeted quantitative real-time polymerase chain reaction for detection of Akebiae Caulis in the traditional Chinese medical formula Longdan Xiegan Wan.

Background: Akebiae Caulis (Mu Tong) is commonly misused by Aristolochiae Manshuriensis Caulis (Guan Mutong) and Clematidis Armandii Caulis (Chuan Mutong), which are nephrotoxic and carcinogenic. However, in the Pharmacopoeia of the People's Republic of China (2015 Edition), the method for determining Akebiae Caulis remains undefined. Methods: We used DNA barcode-based next-generation sequencing (NGS) combined with quantitative real-time polymerase chain reaction (qPCR) to detect Akebiae Caulis in Longdan Xiegan Wan (LDXGW) for the first time. Compared with chromatographic studies, NGS enables better evaluation of the ingredient components of traditional Chinese medicine (TCM) preparations. The feasibility of qPCR using species-specific primers to determine the authenticity of species has been validated. In this study, the constituents of Akebiae Caulis in LDXGW from three different manufacturers were scanned by NGS. The independently developed qPCR detection primers of Akebiae Caulis, Aristolochiae Manshuriensis Caulis, and Clematidis Armandii Caulis were specifically used to analyze the LDXGW mentioned above. Results: The results showed that qPCR detected Clematidis Armandii Caulis in all commercial samples. Meanwhile, NGS detected the counterfeit species Clematis peterae (Tie-Xian Lian) in all samples. We found that qPCR shows a difference in detecting Akebiae Caulis, but it was not able to identify the unknown additives and adulterants for the primer pairs of Clematidis Armandii Caulis. Conclusions: Hence, it is sensitive and rapid, qPCR is not suitable for detection alone. The NGS approaches offer important novel insights that complement the qPCR method. The combination of NGS and qPCR will be a powerful complement to traditional identification methods of TCM substances.

Epimutation of MMACHC compound to a genetic mutation in cblC cases.

BACKGROUND: Methylmalonic aciduria (MMA) combined with homocystinuria, cobalamin(cbl)C deficiency type (OMIM 277400), is the most common autosomal recessive inherited disorder of intracellular cobalamin metabolism caused by mutations in the MMACHC gene (OMIM 609831), of which more than 100 mutations have been identified to date. In this study, we only identified a coding mutation in one allele at the MMACHC gene locus, and no large fragments deletion or duplication were found. Up to now, only three epimutation cblC cases were reported. We hypothesized whether the MMACHC was hypermethylated. METHODS: To address this hypothesis, the entire coding region and adjacent splice sites of the panel genes involved in metabolic diseases were sequenced using the Illumina HiSeq X platform, followed by confirmation via Sanger sequencing in their parents and brothers. Methylation analysis of the MMACHC was performed using an EpiTect Bisulfite Kit and methylation-specific PCR (MSP) to investigate the role of epimutations in cblC disease. RESULTS: We identified a clearly pathogenic single heterozygous c.658_660del, p. (K220del) mutation, which was also identified in the mother. Analysis of the MMACHC indicated a heterozygous epimutation consisting of 34 hypermethylated CpG sites in a CpG island encompassing the promoter and first exon of the MMACHC, which was also identified in the father. Furthermore, we identified a single heterozygous c.*2C>T mutation in the sixth exon of the PRDX1 (OMIM 176763) in patients and their fathers, which was the only sequence variation that segregated with the MMACHC methylation. Neither c.658_660del and epimutation in MMACHC nor c.*2C>T in PRDX1 was discovered in her brother. CONCLUSION: We report compound heterozygotes in MMACHC for a genetic mutation and an epimutation in cblC cases. To our best knowledge, this is the first report of two cblC cases from China caused by compound heterozygous mutations with a coding mutation in one allele and an epimutation in the other at the MMACHC locus.