Identification Strategy of Biological Half Sibling Relationship.

OBJECTIVES: To compare the application value of the likelihood ratio (LR) method and identity by state (IBS) method in the identification involving half sibling relationships, and to provide a reference for the setting of relevant standards for identification of half sibling relationship. METHODS: (1) Based on the same genetic marker combinations, the reliability of computer simulation method was verified by comparing the distributions of cumulated identity by state score (CIBS) and combined full sibling index in actual cases with the distributions in simulated cases. (2) In different numbers of three genetic marker combinations, the simulation of full sibling, half sibling and unrelated individual pairs, each 1 million pairs, was obtained; the CIBS, as well as the corresponding types of cumulative LR parameters, were calculated. (3) The application value of LR method was compared with that of IBS method, by comparing the best system efficiency provided by LR method and IBS method when genetic markers in different amounts and of different types and accuracy were applied to distinguish the above three relational individual pairs. (4) According to the existing simulation data, the minimum number of genetic markers required to distinguish half siblings from the other two relationships using different types of genetic markers was estimated by curve fitting. RESULTS: (1) After the rank sum test, under the premise that the real relationship and the genetic marker combination tested were the same, there was no significant difference between the simulation method and the results obtained in the actual case. (2) In most cases, under the same conditions, the system effectiveness obtained by LR method was greater than that by IBS method. (3) According to the existing data, the number of genetic markers required for full-half siblings and half sibling identification could be obtained by curve fitting when the system effectiveness reached 0.95 or 0.99. CONCLUSIONS: When distinguishing half sibling from full sibling pairs or unrelated pairs, it is recommended to give preference to the LR method, and estimate the required number of markers according to the identification types and the population data, to ensure the identification effect.

Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis.

AIM: The promoter of human interleukin-10 (IL10), a cytokine crucial for suppressing inflammation and regulating immune responses, contains an interspecies-conserved sequence with CpG motifs. The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA). METHODS: Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human, macaque and mouse IL10 genes. Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected. The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 µmol/L). The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA, respectively. The methylation of CpGs in the IL10 promoter was determined by pyrosequencing. Chromatin immunoprecipitation (ChIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions. RESULTS: One interspecies-conserved sequence was found within the IL10 promoter. The upstream CpGs at -408, -387, -385, and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls. In contrast, the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016), which was correlated with higher IL10 mRNA and serum levels. In the 5-azacytidine-treated PBMCs, the CpG motifs were demethylated, and the expression levels of IL10 mRNA and protein was significantly increased. CHIP assays revealed increased phospho-CREB binding to the IL10 promoter. CONCLUSION: The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.

[Genetic polymorphisms of nine non-DNA combined index system short tandem repeat loci in Hebei Han population and application in paternity testing].

OBJECTIVE: To investigate the polymorphisms of 9 non-DNA combined index system (CODIS) short tandem repeats (STRs), i.e., D7S3048, D8S1132, D11S2368, D2S1772, D6S1043, D13S325, D12S391, GATA198B05, D18S1364 in Hebei Han population, and evaluate the usage of them in paternity testing. METHODS: One hundred and forty-seven unrelated healthy individuals from the Han population of Hebei province were genotyped using STRtyper10G kit including 9 STR loci on ABI 3130 Genetic Analyzer. Hardy-Weinberg equilibrium and population genetic parameters were calculated. Fourteen cases of motherless paternity testing and 2 cases of standard trios with mutation in 1 locus were detected using STRtyper10G. RESULTS: (1) Ninety-nine alleles and 336 genotypes were observed in the 9 STR loci in the population. The cumulative discrimination power(DP) was higher than 0.999,999,999. The cumulative probability of exclusion (PE) for trios and duos were 0.999,974 and 0.998,759 respectively. Departure from Hardy-Weinberg equilibrium was not observed in any of the 9 loci. (2) The combined paternity index (PI) of the 14 cases of motherless paternity testing ranged from 10³-106 for 15 STR loci in ID, whereas it reached 105-109 for 22 independent STR loci included in ID and STRtyper 10G. Possible mutation in FGA and vWA was observed in 2 cases of trios, and the combined PI was 5945 and 1840 respectively for 15 STR loci in ID. Adding STRtyper 10G to detect these 2 cases, the combined PI reached 2.76 × 107 and 4.88 × 107 respectively. CONCLUSION: The genetic polymorphism of the 9 non-CODIS STR loci included in STRtyper 10G was quite high in Chinese Hebei Han population, indicating the 9 STR loci are valuable as complement markers for ID and PP16 kit in motherless paternity testing, paternity testing with mutation and other kinds of complicated paternity testing.

Cholecystokinin octapeptide regulates lipopolysaccharide-activated B cells co-stimulatory molecule expression and cytokines production in vitro.

Cholecystokinin octapeptide (CCK8) can exert the immunoregulatory roles through activating immune cell surface receptors such as T lymphocytes, macrophages, dendritic cells, and so on. In this study, we discussed the effects of CCK8 on lipopolysaccharide (LPS)-activated B cells in terms of the expression of co-stimulatory molecules, and the capacity to activate CD4(+) T cells and cytokines production in vitro. The results revealed that B cells expressed two types of CCK receptors; CCK8 inhibited the expression of co-stimulatory molecules such as CD80 and CD86 on LPS-activated B cells, suppressed the proliferation of allogeneic T cells in a dose-dependent manner, and also reduced the secretion of Th1-type cytokine IFN-γ, whereas enhanced the secretion of Th2-type cytokine IL-4 by LPS-activated B cells. Both CCK1R and CCK2R participated in these effects. Taken together, CCK8 is capable of exerting immunomodulatory functions through B cells.

[Genetic polymorphism of four X chromosome short temden repeats loci in Hebei Han population].

OBJECTIVE: To investigate the polymorphism of loci DXS6800, DXS6797, GATA172D05, DXS986 four loci in Hebei Han population. METHODS: The genome DNA of unrelated individuals,the families and rotten materials were extracted with phenol-chloroform method and Chelex-100 method,respectively. The PCR products were detected by the polyacrylamide gel electrophoresis and DNA sequencing analysis. RESULTS: Among 150 unrelated males and 150 unrelated females from Hebei Han population, 25 alleles were found in the 4 loci. One hundred and thirty-eight haplotypes of the male were detected. The haplotype diversity reached 0.9986. CONCLUSION: The findings provided the polymorphic data of DXS6800, DXS6797, GATA172D05, and DXS986 loci in Hebei Han population. The four loci are relatively abundant in polymorphic information for identification and the obtained data of Hebei Han population can be applied to the X-STR genetic data bank.

[Methylation status of the IL-10 gene promoter in the peripheral blood mononuclear cells of rheumatoid arthritis patients].

Interleukin 10(IL-10), as an immunoregulatory cytokine, plays an important role in rheumatoid arthritis (RA). IL-10 gene silencing is associated with the chromatin remodeling in differentiated Th1 and Th2 cells. To explore the relationship between IL-10 promoter methylation and gene silencing in the pathogenesis of RA, IL-10 mRNA, protein expression and promoter methylation status were analyzed in the peripheral blood mononuclear cells (PBMC) of 34 RA patients and 30 healthy controls by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and methylation specific polymerase chain reaction (MSP), respectively. The results showed that IL-10 mRNA and protein expression in RA patients seemed to be lower than that in healthy controls, but there was no statistically significant difference (P>0.05). IL-10 promoter was methylated at a frequency of 85.29% in RA cases, which was significantly higher than the percentage in healthy controls (43.33%) (c 2 =12.439, P=0.000). IL-10 promoter methylation and mRNA expression showed a strong negative correlation (r=-0.579, P=0.001). IL-10 promoter methylation, but not mRNA expression, also correlated statistically with the number of arthritic joints. However, there were no statistical correlations between IL-10 promoter methylation (or mRNA expression) and clinical indices of RA, such as the levels of erythrocyte sedimentation rate (ESR), C reactive protein (CRP) and rheumatic factor (RF) or age (P>0.05). These findings suggest that promoter methylation may be a crucial mechanism of IL-10 gene inactivation in RA and IL-10 promoter CpG island hypermethylation might be involved in the occurrence and development of RA.

[Polymorphism of five short tandem repeat loci on chromosome X in Hebei Han population].

OBJECTIVE: To investigate the polymorphism of DXS6801, DXS6809, DXS7423, DXS7424, DXS9902 five loci in Hebei Han population. METHODS: The PCR products were detected by the polyacrylamide gel electrophresis and DNA sequencing analysis. RESULTS: Among 114 irrelative males and 118 irrelative females from Hebei Han population, 31 alleles were found in the 5 loci. One hundred and one haplotypes of the male were detected and the haplotype diversity reached 0.9975. CONCLUSION: The five loci are relatively abundant in polymorphic information for identification and paternity test. And the obtained data of Hebei Han population can be applied to the X-short tandem repeat genetic data bank.

[Polymorphic analysis of four Y short tandem repeat loci in Han nationality of Hebei province in China].

OBJECTIVE: To investigate the distribution of the polymorphism of the Y-chromosomal loci DYS438, DYS439, GATA A7.1 and GATA A7.2 among Han population in Hebei province. METHODS: With the use of PCR followed by polyacrylamide gel electrophoresis and silver staining, the allele frequencies of these loci in 164 unrelated men of Han population were investigated. RESULTS: Four, five, five, four alleles were observed at the loci DYS438, DYS439, GATA A7.1 and GATA A7.2 respectively; the frequencies of these alleles ranged from 0.0359 to 0.6587, from 0.0179 to 0.4107, from 0.0122 to 0.4146 and from 0.0476 to 0.5238 respectively; the probability discrimination of these loci were 0.5121, 0.6811, 0.6679 and 0.6327 respectively. Seventy different haplotypes were found at these loci. Thirty-six different haplotypes appeared only once. The power of discrimination of these four loci was 0.9480. CONCLUSION: The results demonstrate that these loci(DYS438, DYS439, GATA A7.1 and GATA A7.2) are good genetic markers with high determination power and can be applied to individual identification, especially in paternity test and the detection of mixed samples.

[Sequence polymorphism of mtDNA HV1, HV2 overlapping fragments and coding region 8430-8673nt in Han population of Hebei province].

OBJECTIVE: To investigate the sequence polymorphism of mtDNA HV1,HV2 overlapping fragments and coding region encompassing position 8430-8673 in Hebei Han population. METHODS: Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with sequencing method was used to detect the haplotype distribution of mtDNA in 100 Hebei Han individuals. RESULTS: Ninety-one haplotypes were noted in 100 unrelated individuals. The gene diversity is 0.9985 and the random match probability is 0.0115. Compared with the Anderson sequence, 65 sites of different nucleotide sequences were noted, of which 44 sites were previously registered in MITOMAP, 12 sites were not registered and the gene mutations were different from MITOMAP at 9 positions. CONCLUSION: The obtained data suggest that these loci are valuable genetic markers for personal identification and thus could be used as basic data for the forensic application of mtDNA in Hebei province.