Phosphorylation of Neurofilament Light Chain in the VLO Is Correlated with Morphine-Induced Behavioral Sensitization in Rats.

Neurofilament light chain (NF-L) plays critical roles in synapses that are relevant to neuropsychiatric diseases. Despite postmortem evidence that NF-L is decreased in opiate abusers, its role and underlying mechanisms remain largely unknown. We found that the microinjection of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) into the ventrolateral orbital cortex (VLO) attenuated chronic morphine-induced behavioral sensitization. The microinjection of TSA blocked the chronic morphine-induced decrease of NF-L. However, our chromatin immunoprecipitation (ChIP)-qPCR results indicated that this effect was not due to the acetylation of histone H3-Lysine 9 and 14 binding to the NF-L promotor. In line with the behavioral phenotype, the microinjection of TSA also blocked the chronic morphine-induced increase of p-ERK/p-CREB/p-NF-L. Finally, we compared chronic and acute morphine-induced behavioral sensitization. We found that although both chronic and acute morphine-induced behavioral sensitization were accompanied by an increase of p-CREB/p-NF-L, TSA exhibited opposing effects on behavioral phenotype and molecular changes at different addiction contexts. Thus, our findings revealed a novel role of NF-L in morphine-induced behavioral sensitization, and therefore provided some correlational evidence of the involvement of NF-L in opiate addiction.

Screening of Genes Co-Associated with Sudden Infant Death Syndrome and Infectious Sudden Death in Infancy and Bioinformatics Analysis of Their Regulatory Networks.

OBJECTIVES: The common differentially expressed mRNAs in brain, heart and liver tissues of deceased sudden infant death syndrome (SIDS) and infectious sudden death in infancy (ISDI) confirmed by autopsy was screened by bioinformatics to explore the common molecular markers and pathogenesis of SIDS and ISDI. METHODS: The datasets of GSE70422 and GSE136992 were downloaded, the limma of R software was used to screen differentially expressed mRNA in different tissue samples of SIDS and ISDI decedents for overlapping analysis. The clusterProfiler of R software was used to conduct gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The protein-protein interaction (PPI) network was constructed by STRING database, while the hub gene was screened by cytoHubba plug-in. RESULTS: Compared with the control group, there were 19 significant differentially expressed genes in the tissue samples of SIDS and ISDI decedents, among which 16 in the heart tissue and 3 in the liver tissue, and the astrotactin 1 (ASTN1) gene expression difference in the heart tissue was most significant. The PPI network identified Ras homolog family member A (RHOA), integrin subunit alpha 1 (ITGA1), and H2B clustered histone 5 (H2BC5) were hub genes. The analysis of GO and KEGG showed that differentially expressed genes were enriched in the molecular pathways of actin cytoskeleton regulation, focal adhesion and response to mycophenolic acid. CONCLUSIONS: ASTN1, RHOA and ITGA1 may participate in the development of SIDS and ISDI. The enrichment of differentially expressed genes in immune and inflammatory pathways suggests a common molecular regulatory mechanism between SIDS and ISDI. These findings are expected to provide new biomarkers for molecular anatomy and forensic identification of SIDS and ISDI.

Age estimation using pulp/enamel volume ratio of impacted mandibular third molars measured on CBCT images in a northern Chinese population.

The present study aims to evaluate the relation between chronological age and the ratio of pulp volume (PV) to enamel volume (EV) of impacted mandibular third molars (IMTMs) by using cone-beam computed tomography (CBCT) images and an improved 3D image segmentation technique. A sample of CBCT images of IMTM was collected from 414 northern Chinese subjects (214 male and 200 female clinical patients) ranging in age from 20 to 65 years. The GrowCut effect image segmentation (GCEIS) module algorithm was used to calculate the PV and EV from CBCT images. The total sample was divided into a training group and validation group in a ratio of 7 to 3. The PV/EV ratio (PEr) in the training sample was used to develop a mathematical formula for age estimation as follows: age = - 5.817-21.726 × Ln PEr (p < 0.0001) (Ln, natural logarithm). The mean absolute error (MAE) and root mean square error (RMSE) were used to determine the precision and accuracy of the mathematical formula in the validation group and all samples. The MAEs in the male, female, and pooled gender samples were 9.223, 7.722, and 8.41, respectively, and the RMSEs in the male, female, and pooled gender samples were 10.76, 9.58, and 9.986, respectively. The precise and accurate results indicate that the PEr of IMTM in CBCT images is a potential index for dental age estimation and is possible to be used in forensic medicine.

Novel Association between Flavin-Containing Monooxygenase 3 Gene Polymorphism and Antithyroid Drug-Induced Agranulocytosis in the Han Population.

Some effective antithyroid drugs (ATDs) have been widely used for patients with Graves' disease (GD) but are associated with ATD-induced agranulocytosis. We selected 29 ATD-induced agranulocytosis patients, 44 ATD-induced neutropenia patients, and 140 GD controls among the Chinese Han population who were recruited at the First Affiliated Hospital of Xi'an Jiao Tong University. We assessed their response to ATDs treatment by performing genotyping for a candidate gene association study of samples from patients receiving treatment. Human flavin-containing monooxygenase 3 (FMO3), which is the major hepatic enzyme involved in the production of N-oxide of trimethylamine, catalyzes the oxygenation of a variety of drug compounds. Six single SNP, genotype, haplotype (HAP), and association analyses of the FMO3 gene with ATD-induced agranulocytosis/neutropenia under different models (i.e., additive, dominant, and recessive models) were performed. Rs1736557, which caused an amino acid variation V257M, showed a strong association between ATD-induced agranulocytosis and GD controls after Bonferroni correction (p = 0.011, OR 2.301, 95% CI 1.201-4.409). The presence of HAP 3 (HAP3) in the FMO3 gene HAP was statistically associated with ATD-induced agranulocytosis (p = 0.038, permutation p value). Our findings indicate that genetic variations in the FMO3 gene are associated with the response to ATDs maintenance treatment in ATD-induced agranulocytosis patients of -Chinese Han population.

[Clinical Application of Loop-mediated Isothermal Amplification in Detection of Mycoplasma Pneumoniae].

Objective To explore the clinical value of one-step visualization loop-mediated isothermal amplification(LAMP)in the detection of Mycoplasma pneumoniae(Mp). Methods One-step visualized LAMP,polymerase chain reaction(PCR),and enzyme-linked immunosorbent assay(ELISA)were used to simultaneously detect 108 clinical Mp specimens in children,which included 73 cases of Mp infection diagnosed by PCR and 35 cases of other chronic/acute respiratory tract infections.On the first day of admission,one-step visualization LAMP,PCR(fluorimetric method),and ELISA were used to test the throat swab and serum sample obtained from the same patient,and the Kappa value was calculated.The consistence between LAMP and PCR and that between LAMP and ELISA were compared.On the fifth day of admission,40 patients were resampled and the findings of these three tests on the first day and on the fifth day were compared. Results One-step visualization LAMP had a sensitivity of 100% and a specificity of 94.3%,whereas ELISA had a sensitivity of 65.8% and a specificity of 82.9%.The ratio of Kappa camparing one-step visualization LAMP and PCR was 0.956 and the ratio of Kappa camparing one-step visualization LAMP and ELISA was 0.38.The number of positive specimens detected by LAMP was higher than that by ELISA on the first day. Conclusions One-step visualization LAMP has excellent sensitivity and specificity in detecting early acute Mp infection.It has high consistency with PCR and can be applied to detect Mp.

Age estimation in northern Chinese children by measurement of open apices in tooth roots.

The aim of this study was to assess the accuracy of Cameriere's methods on dental age estimation in the northern Chinese population. A sample of orthopantomographs of 785 healthy children (397 girls and 388 boys) aged between 5 and 15 years was collected. The seven left permanent mandibular teeth were evaluated with Cameriere's method. The sample was split into a training set to develop a Chinese-specific prediction formula and a test set to validate this novel developed formula. Following the training dataset study, the variables gender (g), x 3 (canine teeth), x 4 (first premolar), x 7 (second molar), N 0, and the first-order interaction between s and N 0 contributed significantly to the fit, yielding the following linear regression formula: Age = 10.202 + 0.826 g - 4.068x 3 - 1.536x 4 - 1.959x 7 + 0.536 N 0 - 0.219 s [Symbol: see text] N 0, where g is a variable, 1 for boys and 0 for girls. The equation explained 91.2 % (R (2) = 0.912) of the total deviance. By analyzing the test dataset, the accuracy of the European formula and Chinese formula was determined by the difference between the estimated dental age (DA) and chronological age (CA). The European formula verified on the collected Chinese children underestimated chronological age with a mean difference of around -0.23 year, while the Chinese formula underestimated the chronological age with a mean difference of -0.04 year. Significant differences in mean differences in years (DA - CA) and absolute difference (AD) between the Chinese-specific prediction formula and Cameriere's European formula were observed. In conclusion, a Chinese-specific prediction formula based on a large Chinese reference sample could ameliorate the age prediction accuracy in the age group of children.

The influence of impaction to the third molar mineralization in northwestern Chinese population.

Age estimation by third molar mineralization has been developed for a period of time. Recent studies showed that impaction status has an influence on the rate of the third molar mineralization in Europids and male Black African populations. In this regard, the present study was conducted to determine whether the impaction status could delay the chronological process of third molar mineralization in northwestern Chinese population too. A total of 3,512 digital orthopantomograms of 1,255 male and 2,257 female northwestern Chinese subjects aged from 11 to 26 years were assessed based on the formation stages described by Demirjian et al. with two modifications. Mineralization stage and impaction status were determined for all third molars. Statistical measures were calculated at stages C-H in the male and female gender for both impacted and non-impacted third molars. It was ascertained that the mean ages with impacted third molars at stages C-H were 0.02-1.42 years higher in males and 0.04-1.52 years higher in females than those with non-impacted third molars, but statistical differences were only found at stages C, D, and E in males and stages D and E in females. The probabilities of being under 14, 16, or 18 years of age with non-impacted third molars were all higher than those with impacted third molars. The results prove that impacted third molars show significant slower mineralization than non-impacted ones at stages D and E in both males and females of northwestern Chinese population. It is recommended to consider the influence of impaction on the rate of third molar mineralization for dental age estimation.

[Development of CBCT technique and its application on dental age assessment].

Teeth are the hardest organs in the human body. They are not easily affected by external physical and chemical factors to degrade or deform. Dental age assessment has been widely used in forensic practice. Dental image is one of the most common methods in the age estimation. The emergence of cone beam computed tomography (CBCT) technology provides a new way to obtain three-dimensional image of teeth. It has many advantages such as low-dose radiation, short-time scanning, high-precision image, and finical convenience. Recent development of CBCT technology and its application on age estimation are reviewed in this paper.

The 5-HT7 receptors in the VLO contribute to the development of morphine-induced behavioral sensitization in rats.

The 5-hydroxytryptamine 7 receptor (5-HT7R) is one of the most recently cloned serotonin receptors which have been implicated in many physiological and pathological processes including drug addiction. Behavioral sensitization is the progressive process during which re-exposure to drugs intensified the behavioral and neurochemical responses to drugs. Our previous study has demonstrated that the ventrolateral orbital cortex (VLO) is critical for morphine-induced reinforcing effect. The aim of the present study was to investigate the effect of 5-HT7Rs in the VLO on morphine-induced behavioral sensitization and their underlying molecular mechanisms. Our results showed that a single injection of morphine, followed by a low challenge dose could induce behavioral sensitization. Microinjection of the selective 5-HT7R agonist AS-19 into the VLO during the development phase significantly increased morphine-induced hyperactivity. Microinjection of the 5-HT7R antagonist SB-269970 suppressed acute morphine-induced hyperactivity and the induction of behavioral sensitization, but had no effect on the expression of behavioral sensitization. In addition, the phosphorylation of AKT (Ser 473) was increased during the expression phase of morphine-induced behavioral sensitization. Suppression of the induction phase could also block the increase of p-AKT (Ser 473). In conclusion, we demonstrated that 5-HT7Rs and p-AKT in the VLO at least partially contribute to morphine-induced behavioral sensitization.

NMDA receptor-mediated synaptic transmission in prefrontal neurons underlies social memory retrieval in female mice.

Social memory is the ability to discriminate familiar conspecific from the unknown ones. Prefrontal neurons are essentially required for social memory, but the mechanism associated with this regulation remains unknown. It is also unclear to what extent the neuronal representations of social memory formation and retrieval events overlap in the prefrontal cortex (PFC) and which event drives social memory strength. Here we asked these questions by using a repeated social training paradigm for social recognition in FosTRAP mice. We found that after 4 days' repeated social training, female mice developed stable social memory. Specifically, repeated social training activated more cells that were labeled with tdTomato during memory retrieval compared with the first day of memory encoding. Besides, combining TRAP with c-Fos immunostaining, we found about 30% of the FosTRAPed cells were reactivated during retrieval. Moreover, the number of retrieval-induced but not first-day encoding-induced tdTomato neurons correlates with the social recognition ratio in the prelimbic but not other subregions. The activated cells during the retrieval session also showed increased NMDA receptor-mediated synaptic transmission compared with that in non-labeled pyramidal neurons. Blocking NMDA receptors by MK-801 impaired social memory but not sociability. Therefore, our results reveal that repetitive training elevates mPFC involvement in social memory retrieval via enhancing NMDA receptor-mediated synaptic transmission, thus rendering stable social memory.

Single-cell RNA sequencing reveals a novel inhibitory effect of ApoA4 on NAFL mediated by liver-specific subsets of myeloid cells.

The liver immune microenvironment is a key element in the development of hepatic inflammation in NAFLD. ApoA4 deficiency increases the hepatic lipid burden, insulin resistance, and metabolic inflammation. However, the effect of ApoA4 on liver immune cells and the precise immune cell subsets that exacerbate fatty liver remain elusive. The aim of this study was to profile the hepatic immune cells affected by ApoA4 in NAFL. We performed scRNA-seq on liver immune cells from WT and ApoA4-deficient mice administered a high-fat diet. Immunostaining and qRT-PCR analysis were used to validate the results of scRNA-seq. We identified 10 discrete immune cell populations comprising macrophages, DCs, granulocytes, B, T and NK&NKT cells and characterized their subsets, gene expression profiles, and functional modules. ApoA4 deficiency led to significant increases in the abundance of specific subsets, including inflammatory macrophages (2-Mφ-Cxcl9 and 4-Mφ-Cxcl2) and activated granulocytes (0-Gran-Wfdc17). Moreover, ApoA4 deficiency resulted in higher Lgals3, Ctss, Fcgr2b, Spp1, Cxcl2, and Elane levels and lower Nr4a1 levels in hepatic immune cells. These genes were consistent with human NAFLD-associated marker genes linked to disease severity. The expression of NE and IL-1ß in granulocytes and macrophages as key ApoA4 targets were validate in the presence or absence of ApoA4 by immunostaining. The scRNA-seq data analyses revealed reprogramming of liver immune cells resulted from ApoA4 deficiency. We uncovered that the emergence of ApoA4-associated immune subsets (namely Cxcl9+ macrophage, Cxcl2+ macrophage and Wfdc17+ granulocyte), pathways, and NAFLD-related marker genes may promote the development of NAFL. These findings may provide novel therapeutic targets for NAFL and the foundations for further studying the effects of ApoA4 on immune cells in various diseases.

[Epigenetic mechanisms of the influence of maternal care on off spring development].

It has been demonstrated that the social environment early in life has a long lasting effect on the physical and psychological health of the human body. However, understanding of the relationship between early life experiences, such as maternal care behavior, and life-long cognitive and emotional health can only rely on the studies on animal models. In this paper, we summarized the maternal care effects on both defensive responses to stress and reproductive behavior in rat, and explored the possible underlying epigenetic mechanisms for these effects. Based on this model, we further investigated the significance of such epigenetic effects on human mental health.

Atorvastatin improves bone marrow endothelial progenitor cell function from patients with immune-related hemocytopenia.

BACKGROUND: Immune-related hemocytopenia (IRH) is a type of autoimmune disease that targets bone marrow hematopoietic cells. This study investigated the influence of atorvastatin on the functional aspects of bone marrow endothelial progenitor cells (BM EPCs) in IRH patients. METHODS: BM EPCs were isolated from 15 patients with IRH and 20 normal controls. The isolated BM EPCs were characterized by flow cytometry. Cell counting kit-8, flow cytometry, and Transwell migration assays were used to determine the proliferation, apoptosis, and migration of BM EPCs, respectively. Protein levels were determined by western blot assay. RESULTS: The BM EPCs isolated from IRH patients showed reduced proliferation, increased apoptosis, and attenuated migratory ability compared to those from normal controls. Western blot analysis showed that the protein level of p-p38 was significantly increased, while that of Phosphorylated protein kinase B (p-AKT) was significantly decreased in the BM EPCs from IRH patients, compared to BM EPCs from healthy subjects. Cell proliferation and migration were significantly enhanced by atorvastatin, recombinant human thrombopoietin, and SB20358 compared to the untreated BM EPCs from IRH patients. Atorvastatin, Recombinant human thrombopoietin (TPO), and SB20358 treatment significantly suppressed the protein levels of p-p38 protein, but increased those of p-AKT in BM EPCS from IRH patients. CONCLUSIONS: In summary, atorvastatin increases the number and function of BM EPCs in IRH patients by regulating the p38 and AKT signaling pathways.

Association between a TCF4 Polymorphism and Susceptibility to Schizophrenia.

The basic helix-loop-helix (bHLH) transcription factor 4 (TCF4) had been identified as a susceptibility gene associated with schizophrenia (SCZ) by GWAS, but inconsistent results have been found in other studies. To validate these findings and to reveal the effects of different inheritance models, rs2958182, rs1261085, rs8766, and rs12966547 of the TCF4 gene were genotyped in the Northwest Han Chinese population (448 cases and 628 controls) via a multiplex polymerase chain reaction SNPscan assay. Single SNP, genotype, and association analyses with three different models were performed. We observed genotype and allele distributions of four SNPs that showed nonsignificant associations in the Northwest Han Chinese population. However, published datasets (51,892 cases and 68,498 controls) were collected and combined with our experimental results to ascertain the association of the TCF4 gene SNPs and SCZ, which demonstrated that rs2958182 (P=0.003) was a significant signal based on a systematic meta-analysis. To clarify the biological role of rs2958182, it is important to improve the understanding of the pathophysiology of SCZ.

Tyrosine Hydroxylase Gene Polymorphisms Contribute to Opioid Dependence and Addiction by Affecting Promoter Region Function.

Mounting evidence shows that drug dependence involves the complex interplay between genetics and the environment. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) synthesis, which plays an essential role in the development of drug addiction. Noradrenergic dysfunction due to abnormalities TH expression has been implicated in the pathogenesis of drug addiction. We profiled thirteen single-nucleotide polymorphisms (SNPs) and one VNTR (TCAT repeat, UniSTS:240,639) in 512 cases and 600 healthy Chinese subjects to evaluate the relationship between common variants within the TH gene and opioids dependence (OD) in the Chinese Han population. The single-marker analysis determined that rs10770141 (p < 0.001, OR 1.739, 95% CI 1.302 - 2.323) and rs10770140 (p = 0.002, OR 1.536, 95% CI 1.164 - 2.026) are risk variants for OD. The haplotype-association analyses determined that A-C-C-C was a risk factor (p = 0.006, OR 1.662, 95% CI 1.241 - 2.225) for OD. We also observed a significant association between (TACT)9/9 and the duration of transition from the first time using opioids to the development of opioid dependence (DTFUD) (p = 0.002, OR 2.153, 95% CI 1.319 - 3.513). Taken together, this study suggests that TH gene polymorphisms may contribute to the risk of OD in the Chinese Han population.

The pronociceptive role of 5-HT6 receptors in ventrolateral orbital cortex in a rat formalin test model.

Recent studies have shown the 5-HT6 receptors are expressed in regions which are important in pain processing such as the cortex, amygdala, thalamus, PAG, spinal cord and dorsal root ganglia (DRG), suggesting a putative role of 5-HT6 receptors in pain modulation. The ventrolateral orbital cortex (VLO) is part of an endogenous analgesic system, consisting of the spinal cord - thalamic nucleus submedius (Sm) - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study assessed the possible role of 5-HT6 receptors in the VLO in formalin-induced inflammatory pain model. Firstly we found that microinjection of selective 5-HT6 receptor agonists EMD-386088 (5 µg in 0.5 µl) and WAY-208466 (8 µg in 0.5 µl) both augmented 5% formalin-induced nociceptive behavior. Microinjection of selective 5-HT6 receptor antagonist SB-258585 (1,2 and 4 µg in 0.5 µl) significantly reduced formalin-induced flinching. Besides, the pronociceptive effects of EMD-386088 and WAY-208466 were dramatically reduced by SB-258585, implicating 5-HT6 receptor mechanisms in mediating these responses. In addition, the pronociceptive effect of EMD-386088 was also prevented by the adenylate cyclase (AC) inhibitor SQ-22536 (2 nmol in 0.5 µl) and the protein kinase A (PKA) inhibitor H89 (10 nmol in 0.5 µl), respectively. We further confirmed the above results with quantification of spinal c-fos expression. Taken together, our results suggested that 5-HT6 receptors play a pronociceptive role in the VLO in the rat formalin test due to its activation of AC - PKA pathway. Therefore, cerebral cortical 5-HT6 receptors could be a new target to develop analgesic drugs.

Cue-elicited drug craving represses ERK activation in mice prefrontal association cortex.

Morphine addiction is characterized by compulsive drug-taking behavior and high rates of relapse that reflect reward-controlled learning, consolidation and reconsolidation of drug cues. Extracellular signal-regulated protein kinase (ERK) is one of the cellular molecules that have been highly implicated in the synaptic plasticity processes of learning and memory in cocaine addiction. However, the roles of ERK in the morphine-paired conditioned place preference (CPP) are not clear. In the present study, we found that compared to the morphine-unpaired and saline-paired and saline-unpaired groups, morphine-paired mice showed depressed ERK2 activity in the Frontal Association Cortex (FrA), whereas ERK1 activity was not changed in the same region. In the Accumbens Nucleus (Acb) and Caudate Putamen (CPu) that are associated with cocaine addiction, the activities of ERK1 and ERK2 among four groups showed no difference. These results suggest that the FrA plays an important role in morphine craving and that ERK2 is involved in eliciting the environment-related morphine craving, which is totally different from those induced by morphine itself.

[Gene expression profiling in drug addicted brain].

Drug addiction, a chronic brain disease caused by interaction of in vitro drug toxification and in vivo gene susceptibility, has been widely studied but its underlining mechanism is so far been elucidated. A major goal in this field is to identify drug-induced molecular changes and their effects on brain function. By the advance of high throughput technologies in genomics and transcriptomics, the whole gene expression profile in addicted brain could be obtained and proved to be a very powerful tool to unclose the molecular mechanism underlying the addiction biology context. Here, we summarized the progress of serial analysis of gene expression (SAGE) and microarray, as well as their application in drug addiction.

[Sequence polymorphism of mtDNA HVR Iand HVR II of Oroqen ethnic group in Inner Mongolia].

Venous blood samples from 50 unrelated Oroqen individuals living in Inner Mongolia were collected and their mtDNA HVR I and HVR II sequences were detected by using ABI PRISM377 sequencers. The number of polymorphic loci, haplotype, haplotype frequence, average nucleotide variability and other polymorphic parameters were calculated. Based on Oroqen mtDNA sequence data obtained in our experiments and published data, genetic distance between Oroqen ethnic group and other populations were computered by Nei's measure. Phylogenetic tree was constructed by Neighbor Joining method. Comparing with Anderson sequence, 52 polymorphic loci in HVR I and 24 loci in HVR II were found in Oroqen mtDNA sequence, 38 and 27 haplotypes were defined herewith. Haplotype diversity and average nucleotide variability were 0.964+/-0.018 and 7.379 in HVR I, 0.929+/-0.019 and 2.408 in HVR II respectively. Fst and dA genetic distance between 12 populations were calculated based on HVR I sequence, and their relative coefficients were 0.993(P < 0.01). A phylogenetic tree was constructed based on genetic distances and included Oroqen, Taiwan and South Han population in a clade, which indicated near genetic relation between them, and far relation with northern Han, Mongolian and other foreign populations. The genetic polymorphism of mtDNA HVR I and HVR II in Oroqen ethnic group has some specificities compared with that of other populations. These data provide a useful tool in forensic identification, population genetic study and other research fields.

[Detection of ABO genotype genetic polymorphism by multiplex-PCR based sequencing and application in forensic medicine].

Multiplex PCR-direct sequencing method was established to detect 9 different SNPs in exon 6 and exon 7 of ABO genes and could identify at least 28 different ABO genotypes. Population study was carried out in a sample of 80 unrelated Chinese Tibetan minority individual dwelled in Qinghai Province. The method was also applied to forensic cases. A variety of degeneration forensic samples, including blood stain, hair root, swab, bone and mixed stain were successfully identified by this efficient method and in conformance with serological typing. There were no significant deviations from Hardy-Weinberg equilibrium in ABO genotypes of Tibetan population. The heterozygosity, polymorphic information content, discrimination power, paternity of exclusion, and probability of genetic identity were 0.675, 0.672, 0.874, 0.391, and 0.126 respectively. The gene frequency of ABO was O>B>A. The multiplex PCR-directed sequencing method can accurately and reliably detect ABO genotypes in many kinds of samples, and it improves personal identification efficiency. The ABO genotype is high variance in Qinghai Tibetan minority group, and it can be applied in forensic medicine and population genetic study.