Decreased ALCAM expression and promoter hypermethylation is associated with preeclampsia.

Preeclampsia (PE) is a major obstetrical complication that results in maternal and fetal morbidity and mortality. Aberrant epigenetic modifications are widely involved in the pathogenesis of PE. Previously, the activated leukocyte cell adhesion molecule (ALCAM) was reported to be required for blastocyst implantation but has not been described in the context of pathological pregnancy. This study explored the expression of ALCAM and its methylation levels in the placentas and peripheral venous blood of patients with PE from a Chinese Han population. The mRNA and protein expression levels of ALCAM were downregulated in the PE placentas compared with the control placentas (P < 0.05). The methylation rate of the ALCAM gene promoter was considerably elevated in the placentas (P = 0.003, odds ratio (OR) = 0.264, 95% confidence interval (95% CI) [0.108-0.647], cases n = 47, controls n = 53) and peripheral blood (P = 0.007, OR = 0.455, 95% CI [0.256-0.806], cases n = 100, controls n = 100) of the PE patients compared with those of the normotensive women, suggesting a negative relationship between ALCAM methylation and gene transcription. Moreover, the transcriptional expression of ALCAM was dramatically increased by demethylating treatment in trophoblastic cells. ALCAM is expected to be involved in the pathogenesis of PE through methylation regulation.

[TUBB1 mutation in children with congenital hypothyroidism and thyroid dysgenesis in Shandong, China].

OBJECTIVE: To study the types and characteristics of TUBB1 mutation in children with congenital hypothyroidism (CH) and thyroid dysgenesis (TD) in Shandong, China. METHODS: Mutations of the whole coding region of the TUBB1 gene were analyzed for 289 children with CH and TD in Shandong. Whole-genome DNA was extracted from peripheral blood leukocytes. PCR multiplication was performed for the whole coding region of the TUBB1 gene. Sanger sequencing was performed for the PCR products, and a biological information analysis was performed. RESULTS: Among the 289 children with CH and TD, 4 (1.4%) were found to have a c.952C>T(p.R318W) heterozygous mutation in the TUBB1 gene, resulting in the change of tryptophan into arginine at codon 318 of TUBB1 protein. This mutation was evaluated as "potentially pathogenic" based on the classification criteria and guidelines for genetic variation by American College of Medical Genetics and Genomics. CONCLUSIONS: A novel mutation is detected in the exon of the TUBB1 gene in children with CH and TD in Shandong, suggesting that the TUBB1 gene may be a candidate pathogenic gene for CH children with TD.

Polymorphism of ERCC1 rs3212986 in Chinese Han women with preeclampsia.

OBJECTIVE: To investigate the association between polymorphism of rs3212986 in ERCC1 and susceptibility to preeclampsia in the Chinese Han population. STUDY DESIGN: Samples of 642 preeclampsia patients and 877 controls were genotyped for rs3212986 using TaqMan allele discrimination assays. The genetic and allelic distributions between the groups were compared by Pearson's χ2 test. RESULT: There was no difference in the genotypic and allelic distributions between cases and controls (P>0.05). Statistical difference in genotypic frequencies of rs3212986 was observed between early-onset and late-onset preeclampsia (χ2=6.985, P=0.030). When subdivided into TT/GG+GT groups, a significant difference was found between early-onset and late-onset preeclampsia (χ2=6.528, P=0.011, OR=2.011, 95%CI 1.167-3.465). CONCLUSION: The polymorphisms of rs3212986 showed no association with the risk of preeclampsia in the Chinese Han population. However, the difference in the genotypic distribution between early-onset and late-onset preeclampsia suggest the need for future studies.

[Toxoplasma gondii Infection in HIV-infected Individuals in Henan Province].

Genomic DNA was extracted from 1 038 peripheral blood samples from HIV-infected individuals in Henan Province. One-step single-tube nested PCR was performed to amplify the 529 bp repeating sequences of Toxoplasma gondii. Of the 1 038 samples （762 from males and 276 from females）, 66 showed positive PCR results, with a positive rate of 6.4%. The PCR positive rate in males and females was 6.3% （48/762） and 6.5% （18/276） respectively. The PCR positive rate in the married HIV individuals was 4.9%（25/508）, and that in unmarried, divorced and widowed HIV individuals was 7.7% （41/530）（χ2 = 3.451, P> 0.05）. The PCR positive rate in HIV individuals with a high-school educational level or above was 6.9%（34/489）, and that in those below the high-school level was 5.8% （32/549）（χ2 = 0.545, P> 0.05）. The highest infection rate was in the age group of 20-40 years（7.6%, 31/410）. In addition, the Toxoplasma infection rate in those with and without a history of venereal diseases, and those with an unknown history was 8.0%（9/113）, 6.5%（50/773） and 4.6%（7/152） respectively （χ2 = 0.355, P> 0.05）.

[DUOX2 mutations in children with congenital hypothyroidism].

OBJECTIVE: To study the features of DUOX2 mutations and genotype-phenotype relationship in children with congenital hypothyroidism (CH), in order to provide evidence for gene diagnosis and gene treatment of CH. METHODS: Blood samples were collected from 10 CH children with thyromegaly. Genomic DNA was extracted from peripheral blood leukocytes. All exons of DUOX2 gene were analyzed using PCR and direct sequencing. RESULTS: G3632A mutation in the exon 28 of DUOX2 that may result in arginine to histidine substitution at codon 1211 was found in one patient. T2033C mutation in the exon 17 of DUOX2 that may result in histidine to arginine substitution at codon 678 was found in three patients. They were all heterozygous mutations. CONCLUSIONS: Heterozygous mutations in DUOX2 may affect protein function and cause CH. The relationship between DUOX2 genotypes and clinical phenotypes is unclear and needs further studies.

Polymorphisms in the vitamin D receptor and risk of gout in Chinese Han male population.

Previous studies have showed that patients with gout showed lower serum 25(OH)D levels. As the specific receptor of vitamin D, VDR plays an important role in regulating immune system by combining with vitamin D. In this study, we investigated whether the functional VDR polymorphisms were associated with susceptibility to gout in Chinese Han male population. A total of 504 patients with gout and 523 gout-free controls were recruited from the Affiliated Hospital of the Medical College, Qingdao University. Genotyping of VDR rs11568820, rs2228570 and rs1544410 was performed by TaqMan allele discrimination assays. An association analysis was carried out using the χ(2) test. A genotype-phenotype analysis was also conducted. Our results showed that polymorphisms of rs11568820 and rs1544410 in VDR were associated with gout in Chinese Han male population. The A allele of both rs11568820 and rs1544410 was associated with the risk of gout [P = 0.012 OR 1.251, 95% CI (1.051-1.490); P = 0.006, OR 1.574, 95% CI (1.139-2.175)]. However, there was no statistic significance between rs2228570 and gout (P = 0.186). Our study suggested that the polymorphisms of VDR may be relevant host susceptibility factors for the development of gout in Chinese Han male population. However, further study should be done in a larger size sample and other ethic to test and verify our result.

Functional study of one nucleotide mutation in pri-miR-125a coding region which related to recurrent pregnancy loss.

MicroRNAs (miRNAs) are short non-coding RNAs which modulate gene expression by binding to complementary segments present in the 3'UTR of the mRNAs of protein coding genes. MiRNAs play very important roles in maintaining normal human body physiology conditions, meanwhile, abnormal miRNA expressions have been found related to many human diseases spanning from psychiatric disorders to malignant cancers. Recently, emerging reports have indicated that disturbed miRNAs expression contributed to the pathogenesis of recurrent pregnancy loss (RPL). In this study, we identified a new mutation site (+29A>G, position relative to pre-miR-125a) by scanning pri-miR-125a coding region in 389 Chinese Han RPL patients. This site was co-existed with two polymorphisms (rs12976445 and rs41275794) in patients heterogeneously and changed the predicted secondary structures of pri-miR-125a. Subsequent in vitro analysis indicated that the A>G mutation reduced mature miR-125a expression, and further led to less efficient inhibition of verified target genes. Functional analysis showed that mutant pri-mir-125a can enhance endometrial stromal cells (ESCs) invasive capacity and increase the sensitivity of ESCs cells to mifepristone. Moreover, we further analyzed the possible molecular mechanism by RIP-chip assay and found that mutant pri-mir-125a disturbed the expression of miR-125a targetome, the functions of which includes embryonic development, cell proliferation, migration and invasion. These data suggest that A>G mutation in pri-miR-125a coding region contributes to the genetic predisposition to RPL by disordering the production of miR-125a, which consequently meddled in gene regulatory network between mir-125a and mRNA.

Effect of 5-aza-2'-deoxycytidine combined with trichostatin A on RPMI-8226 cell proliferation, apoptosis and DLC-1 gene expression.

This study was aimed to investigate the effects of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and histone deacetylase inhibitor trichostatin A (TSA) on DLC-1 gene transcription regulation and molecular biological behaviours in the human multiple myeloma RPMI-8226 cells. The cells were treated respectively with 5-Aza-CdR and TSA alone, or the both combination; the cell proliferation and apoptosis, DLC-1 expression, the protein expression of Ras homolog family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) were examined by CCK-8 method, RT-PCR and ELISA, respectively. The results showed that the 5-Aza-CdR and TSA had cell growth inhibitory and apoptosis-inducing effects in dose-dependent manner (P < 0.05). Compared with a single drug (5-Aza-CdR or TSA alone), the effects were significantly enhanced after treatment with the combination of 5-Aza-CdR and TSA (P < 0.05). DLC-1 was weakly expressed in the control group; the treatment with 5-Aza-CdR alone enhanced its re-expression dose-dependently (P < 0.05). Compared with 5-Aza-CdR alone, 5-Aza-CdR plus TSA enhanced DLC-1 re-expression significantly.Compared with the control, 5-Aza-CdR and TSA significantly decreased RhoA and Rac1 protein expression (P < 0.05). It is concluded that 5-Aza-CdR and TSA can effectively reverse DLC-1 expression of RPMI-8226 cells; TSA has a synergistic effect on its re-expression. 5-Aza-CdR and TSA have significant cell growth inhibitory and apoptosis-inducing effects on RPMI-8226 cells. These effects may be related to the inhibition of Rho/Rho kinase signalling pathway.

[Family-based association study of a variable number of tandem repeat polymorphism of DAT1 gene with Tourette syndrome in a Chinese Han population].

OBJECTIVE: To assess the association of a 40 bp variable number of tandem repeat (VNTR) polymorphism within 3 untranslated region of dopamine transporter gene (DAT1) with Tourette syndrome (TS) in a Chinese Han population. METHODS: A total of 160 TS patients and their parents were recruited. The VNTR polymorphism was detected with polymerase chain reaction-VNTR analysis, and its association with TS and its subtypes were assessed through a family-based association study comprising transmission disequilibrium test (TDT) and haplotype relative risk (HRR) analysis. RESULTS: The repeat numbers at the DAT1 40 bp locus were 11, 10, 9, 7.5 and 7 among the patients and their parents, with the most common type being a 10-repeat allele. No significant association was detected between the polymorphism and TS (TDT: X ² = 0.472, df = 1, P = 0.583; HRR: X ² = 0.313, P = 0.576, OR = 0.855, 95%CI: 0.493-1.481). CONCLUSION: Our data suggested that the VNTR polymorphism of DAT1 gene is not associated with susceptibility to TS in Chinese Han population. However, our results are to be validated in larger sets of patients collected from other populations.

[Association between single nucleotide polymorphism of macrophage migration inhibitory factor-rs1007888 and the pathogenesis of gestational diabetes mellitus].

OBJECTIVE: To investigate the association between single nucleotide polymorphism (SNP) of macrophage migration inhibitory factor (MIF) gene-rs1007888 and the pathogenesis of gestational diabetes mellitus (GDM). METHODS: A total of 120 GDM pregnant women (GDM group) and 165 healthy pregnant women (control group) from Affiliated Hospital of Medical College, Qingdao University were recruited from June 2011 to July 2012. Their age, gestational week, height and weight were recorded. The levels of fasting blood glucose (FBG) and fasting insulin (FIN) were determined. Body mass index (BMI), the hemeostasis model assessment-insulin resistance (HOMA-IR) and hemeostasis model assessment-ß cell function (HOMA-ß) were calculated. DNA was extracted from fasting blood samples. SNP of MIF-rs1007888G/A was determined by DNA sequencing.The FBG, FIN, HOMA-IR and HOMA-ß were compared between GDM group and the control group.They were also compared among pregnancies with different genotypes. RESULTS: (1) GDM group had higher FBG, FIN and HOMA-IR levels, but lower HOMA-ß than the control group (all P < 0.05). (2) MIF-rs1007888 SNP genotype frequencies of GG, GA and AA were 37.5%, 45.8% and 16.7%, and the allelic frequencies of G and A were 60.4%, 39.6% in GDM group; However, in the control group, the frequencies of GG, GA and AA were 26.1%, 54.5% and 19.4%, and the allelic frequencies of G and A were 53.3%, 46.7%, respectively.The distributions of MIF genotypes in GDM patients were significantly different from the healthy subjects (P < 0.05).No significant difference of MIF-rs1007888 allele distributions was observed between GDM group and the control group (P > 0.05). (3) The FBG, FIN and HOMA-IR in pregnant women with GG genotype were statistically higher than those with GA or AA genotypes, while HOMA-ß was lower in women with GG genotype (all P < 0.05). CONCLUSIONS: The SNP of MIF rs-1007888 was related to the insulin resistance and pancreatic ß cell function of pregnant women. GG genotype of MIF-rs1007888 might be a genetic susceptible factor in the pathogenesis of GDM.

[Association between severe preeclampsia and single nucleotide polymorphism of macrophage migration inhibitory factors -173G/C].

OBJECTIVE: To investigate whether single nucleotide polymorphism (SNP) of macrophage migration inhibitory factor (MIF) gene -173G/C is associated with severe preeclampsia. METHODS: Totally 124 severe preeclampsia patients and 160 healthy pregnant women (control group) were included in our study who were recruited consecutively from Affiliated Hospital of Qingdao University Medical College between March 2010 and March 2011. The SNP was detected through SYBR Green PCR. The levels of fasting blood glucose (FBG), fasting insulin (FIN), and serum total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL) and light density lipoprotein (LDL) were determined in every participants. The homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. The allele and genotype frequencies between severe preeclampsia patients and control group were compared. The FBG, FIN, body mass index (BMI), HOMA-IR, TC, TG, HDL and LDL in different genotype were compared. RESULTS: (1) The MIF -173G/C SNP genotype frequencies of GG, CG, and CC were 62.1% (77/124), 30.6% (38/124), 7.3% (9/124), the allelic frequencies of G and C were 77.4% (192/248) and 22.6% (56/248), respectively, in severe preeclampsia patients; the MIF -173G/C SNP genotype frequencies of GG, CG, and CC were 64.4% (103/160), 30.6% (49/160), 5.0% (8/160), the allelic frequencies of G and C were 79.7% (255/320) and 20.3% (65/320), respectively, in the control group. No significant differences were observed in the genotypes and allele distributions of MIF -173G/C SNP between the severe preeclampsia patients and control group (all P > 0.05). (2) The severe preeclampsia patients with CG and CC genotypes had higher BMI compared with the GG genotype [(25 ± 4) versus (22 ± 4) kg/m(2); t = 3.96, P < 0.05].(3) The severe preeclampsia patients with CG and CC genotypes had higher FIN level and higher HOMA-IR compared with the GG genotype [(15.7 ± 2.9) versus (13.6 ± 4.0) mmol/L, 3.3 ± 0.5 versus 2.7 ± 0.6; t = 3.17, t = 5.58, all P < 0.05]. (4) There was no significant difference in FBG, TC, TG, HDL and LDL levels in severe preeclampsia patients with different genotypes (all P > 0.05). CONCLUSIONS: The present study suggests that the MIF -173G/C SNP is associated with insulin resistance in severe preeclampsia patients. The CG and CC genotypes increase the degree of insulin resistance, but it is may not associate with susceptibility among severe preeclampsia patients of Han Chinese women.

[DUOX2 gene mutation in patients with congenital goiter with hypothyroidism].

OBJECTIVE: To identify DUOX2 gene mutation in patients with congenital goiter with hypothyroidism. METHOD: Five patients who had transit congenital hypothyroidism with goiter were enrolled. The exons of DUOX2 gene were amplified and sequenced. RESULT: A heterozygous missense mutation C1329T in the exon 10 of the DUOX2 gene was found in one patient, predicted to result in a Tryptophan to Arginine substitution at codon 376. However no mutation was detected in the other patients. CONCLUSION: p.Arg376Trp mutation in DUOX2 was found in newborns of congenital hypothyroidism. The alleles frequency of this mutation may contribute to the function loss of congenital hypothyroidism.

[Correlation of IL-4 and IL-13 gene polymorphisms with asthma and total serum IgE levels].

OBJECTIVE: To investigate the correlation between the single nucleotide polymorphisms (SNP) of +33C/T in the promoter region of IL-4 gene and +1923C/T in intron-3 region of IL-13 gene and the susceptibility of asthma, and to study the impact of these polymorphisms upon total serum IgE levels. METHODS: The 2 polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), in 150 asthmatic subjects (asthma group) and 160 healthy controls (healthy control group) of the Han nationality in Shandong province enrolled from December 2003 to June 2007. The total serum IgE levels were determined by ELISA. RESULTS: The genotype frequencies of CC, CT and TT in +33C/T sites of IL-4 gene were 43% (68/160), 35% (56/160), 22% (36/160) respectively in the controls, and 18% (27/150), 36% (54/150), 46% (69/150) respectively in the asthmatic subjects. The genotype frequencies of CC, CT and TT in +1923C/T sites of IL-13 gene were 41% (66/160), 43% (68/160), 16% (26/160) respectively in the controls, and 21% (31/150), 38% (57/150), 41% (61/150) respectively in the asthmatic subjects. The distribution of genotype in each sites between the 2 groups was significantly different (chi(2) = 27.821, 26.544 respectively, all P < 0.01). The CT and TT genotypes carried higher risks for asthma than CC genotypes (chi(2) = 21.870, 14.206 respectively, all P < 0.01). The total serum IgE levels of CC, CT and TT in +33C/T sites of IL-4 gene were (92 +/- 37), (122 +/- 45), (146 +/- 44) KU/L respectively in the controls, and (179 +/- 40), (294 +/- 51), (341 +/- 80) KU/L respectively in the asthmatic subjects. The total serum IgE levels of CC, CT and TT in +1923C/T sites of IL-13 gene were (85 +/- 31), (102 +/- 38), (144 +/- 49) KU/L respectively in the controls, and (186 +/- 65), (297 +/- 87), (363 +/- 140) KU/L respectively in the asthmatic subjects. In these 2 sites, the total serum IgE level of asthmatics was higher than that of the controls with the same genotype between the 2 groups (t = 4.653, 6.547, 7.754; and 4.673, 6.784, 8.157 respectively, all P < 0.01). The total serum IgE levels of CT, TT genotypes were higher than CC genotypes in the same group (t = 5.748, 6.253 respectively, all P < 0.01). CONCLUSIONS: There was a significant correlation between the polymorphisms of +33C/T sites of IL-4 and +1923C/T sites of IL-13 gene and susceptibility to asthma and increase of the total serum IgE. The IL-4 gene and IL-13 gene may be important candidate genes for asthma.

Clinical and genetic study of a novel mutation in the REEP1 gene.

OBJECTIVE: To examine the gene mutation associated with clinical phenotype from a Chinese kindred with autosomal dominant hereditary spastic paraplegia (ADHSP). METHOD: To perform linkage analysis and mutation detection. For two affected individual of the family, clinical analysis, electrophysiological examination, and MRI of brain and spinal cord were also performed. RESULT: A novel splice-site mutation (REEP1 c417+1g>a) was identified. Central motor conduction time to the first metatarsal interosseus and anterior tibial muscles were clearly prolonged. Thoracic cord atrophy was found from T1 to T10. CONCLUSION: Our study supports that mutations in REEP1 cause ADHSP and demonstrates genetic heterogeneity in ADHSP. Synapse

[Effect of lipid-bound apoA-I cysteine mutants upon lipopolysaccharide-induced endotoxemia in mice].

OBJECTIVE: To determine the anti-inflammatory functions of different cysteine mutants of apolipoprotein A-I recombinant HDLs. METHODS: The authors reconstituted recombinant HDLs (namely rHDL74, rHDL129, rHDL195 and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects upon LPS-induced endotoxemia in mice. RESULTS: At 24 h post-injection, mice receiving rHDL74 [TNF-alpha: (24 +/- 3) pg/ml; IL-1beta: (45 +/- 5) pg/ml] had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) as compared with control mice receiving either saline or rHDLwt [TNF-alpha: (135 +/- 12) pg/ml; IL-1beta: (82 +/- 8) pg/ml, P < 0.05]. Administration of rHDL74 to mice injected with LPS also led to a protection of lung against acute injury and attenuation of endotoxin-induced clinical symptoms in mice as compared with controls injected with LPS only. CONCLUSION: Compared with rHDLwt, rHDL74 exhibits higher anti-inflammation capabilities. And it may be a potential clinical candidate for therapy for endotoxin-induced septic shock.

A novel mutation in EXT2 gene in a Chinese family with hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is an autosomal-dominant disorder characterized by the presence of bony outgrowths on the long bones. In this report, we describe a Chinese family with HME. Linkage analysis and mutation detection were performed. Linkage with the EXT2 was established in this family. A novel mutation, EXT2 c239-244delG, was identified. Mutation analysis in a family with HME allows for genetic counseling and prenatal diagnosis.

Clinical and genetic study of SPG6 mutation in a Chinese family with hereditary spastic paraplegia.

Mutations in the NIPA1 gene cause autosomal dominant hereditary spastic paraplegia (ADHSP). To date, little is known about the relationship between genotype-phenotype correlation. In order to examine the gene mutation associated with the genotype-phenotype of Chinese kindred with ADHSP, linkage analysis and mutation detection were performed. For affected family members, clinical analysis, electrophysiological examination and MRI of the brain and spinal cord were also performed. Every effected patient had a c316 (G106R) mutation in the NIPA1 gene. Neurophysiological examination revealed decreased amplitude of compound muscle action potentials (CMAP) from the tibial and peroneal motor nerves in most patients. Sensory nerve action potential (SNAP) from the tibialis nerve was not elicited in most patients. Central motor conduction time (CMCT) to the abductor pollicis brevis muscle (APB), first metatarsal interosseus muscle (FMI) and anterior tibial muscle (AT) were either absent or clearly prolonged in all patients. Spinal cord MRI showed different levels of atrophy in every affected individual. These lesions present an increased spot or patch signal on transverse axis T2WI and an intense signal of continual longitudinal strip on the anteroposterior axis. Our study supports that mutations in the NIPA1 gene cause ADHSP and further demonstrates genotype-phenotype correlations in SPG6.

[EXT1 and EXT2 mutation identified by denaturing high performance liquid chromatograph in three families with hereditary multiple exostoses].

OBJECTIVE: To develop a new denaturing high performance liquid chromatograph (DHPLC)-based method to screen patients with EXT gene mutation and to study the gene mutation in three families with multiple exostoses. METHODS: All the exons of EXT gene, including the intro-exon boundaries, were amplified by PCR. Linkage analysis and DHPLC screening were carried out to identify the mutations. DNA sequencing was used to confirm the mutations. RESULTS: Two known splice site mutations, IVS2+1 G to A and IVS7+1 G to T, and two SNPs have been detected in EXT2 or EXT1 gene. CONCLUSION: The transversions of IVS2+1 G to A and IVS7+1 G to T in EXT2 gene are suggested to be the disease-causing mutations and the DHPLC is a high throughout, sensitive, simple, quick, economical method to screen gene mutation in hereditary multiple exostosis.