Genome-wide association and Mendelian randomisation analysis among 30,699 Chinese pregnant women identifies novel genetic and molecular risk factors for gestational diabetes and glycaemic traits.

AIMS/HYPOTHESIS: Gestational diabetes mellitus (GDM) is the most common disorder in pregnancy; however, its underlying causes remain obscure. This study aimed to investigate the genetic and molecular risk factors contributing to GDM and glycaemic traits. METHODS: We collected non-invasive prenatal test (NIPT) sequencing data along with four glycaemic and 55 biochemical measurements from 30,699 pregnant women during a 2 year period at Shenzhen Baoan Women's and Children's Hospital in China. Genome-wide association studies (GWAS) were conducted between genotypes derived from NIPTs and GDM diagnosis, baseline glycaemic levels and glycaemic levels after glucose challenges. In total, 3317 women were diagnosed with GDM, while 19,565 served as control participants. The results were replicated using two independent cohorts. Additionally, we performed one-sample Mendelian randomisation to explore potential causal associations between the 55 biochemical measurements and risk of GDM and glycaemic levels. RESULTS: We identified four genetic loci significantly associated with GDM susceptibility. Among these, MTNR1B exhibited the highest significance (rs10830963-G, OR [95% CI] 1.57 [1.45, 1.70], p=4.42×10-29), although its effect on type 2 diabetes was modest. Furthermore, we found 31 genetic loci, including 14 novel loci, that were significantly associated with the four glycaemic traits. The replication rates of these associations with GDM, fasting plasma glucose levels and 0 h, 1 h and 2 h OGTT glucose levels were 4 out of 4, 6 out of 9, 10 out of 11, 5 out of 7 and 4 out of 4, respectively. Mendelian randomisation analysis suggested that a genetically regulated higher lymphocytes percentage and lower white blood cell count, neutrophil percentage and absolute neutrophil count were associated with elevated glucose levels and an increased risk of GDM. CONCLUSIONS/INTERPRETATION: Our findings provide new insights into the genetic basis of GDM and glycaemic traits during pregnancy in an East Asian population and highlight the potential role of inflammatory pathways in the aetiology of GDM and variations in glycaemic levels. DATA AVAILABILITY: Summary statistics for GDM; fasting plasma glucose; 0 h, 1 h and 2h OGTT; and the 55 biomarkers are available in the GWAS Atlas (study accession no.: GVP000001, https://ngdc.cncb.ac.cn/gwas/browse/GVP000001) .

Genetic basis of pregnancy-associated decreased platelet counts and gestational thrombocytopenia.

ABSTRACT: Platelet count reduction occurs throughout pregnancy, with 5% to 12% of pregnant women being diagnosed with gestational thrombocytopenia (GT), characterized by a more marked decrease in platelet count during pregnancy. However, the underlying biological mechanism behind these phenomena remains unclear. Here, we used sequencing data from noninvasive prenatal testing of 100 186 Chinese pregnant individuals and conducted, to our knowledge, the hitherto largest-scale genome-wide association studies on platelet counts during 5 periods of pregnancy (the first, second, and third trimesters, delivery, and the postpartum period) as well as 2 GT statuses (GT platelet count < 150 × 109/L and severe GT platelet count < 100 × 109/L). Our analysis revealed 138 genome-wide significant loci, explaining 10.4% to 12.1% of the observed variation. Interestingly, we identified previously unknown changes in genetic effects on platelet counts during pregnancy for variants present in PEAR1 and CBL, with PEAR1 variants specifically associated with a faster decline in platelet counts. Furthermore, we found that variants present in PEAR1 and TUBB1 increased susceptibility to GT and severe GT. Our study provides insight into the genetic basis of platelet counts and GT in pregnancy, highlighting the critical role of PEAR1 in decreasing platelet counts during pregnancy and the occurrence of GT. Those with pregnancies carrying specific variants associated with declining platelet counts may experience a more pronounced decrease, thereby elevating the risk of GT. These findings lay the groundwork for further investigation into the biological mechanisms and causal implications of GT.

Amplicon sequencing-based carrier screening for 170 monogenic disorders among children with abnormal LC-MS/MS results.

BACKGROUND: Next-generation sequencing (NGS) has been suggested as a second-tier diagnostic test for newborn screening, which could help identify the carrier status of hundreds monogenic disorders with wider spectrum and earlier stage. METHODS: Among the 1087 children (age from 27 min to 14 years old) underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS), 290 individuals who had at least one abnormal value of LC-MS/MS measurements were sent for amplicon sequencing-based carrier screening (targeting 141 genes for 170 monogenic disorders). Multiplex polymerase chain reaction was used for amplicon capture and library preparation, the NextSeq 500 NGS platform (Illumina PE150) was used for sequencing. The identified clinical significant variants were further validated by Sanger sequencing. RESULTS: Only 89 children carry none of clinical significant variants, other 201 individuals carry 1-4 variants in 63 genes (132 types; 317 in total: 171 pathogenic, 37 likely pathogenic, 29 variants of unknown significance, and 80 disease-associated functional polymorphisms). Besides the three missing samples with 4 variants, 91.1 % of identified variants (285 variants in 54 genes) were completely validated by Sanger sequencing. The most common genetic variants were in UGT1A1, GJB2, PAH, G6PD, and SLC25A13 (top 5 genes), which corresponding to Gilbert/Crigler-Najjar symdrome (n = 89), autosomal recessive hearing loss type 1A (n = 58), phenylketonuria (n = 12), glucose-6-phosphate dehydrogenease deficiency (n = 11) and Citrin deficiency (n = 9). More than 42 children present higher phenylalanine in LC-MS/MS, but only 12 of them were identified to carry clinical significant variants in PAH gene. CONCLUSION: The amplicon sequencing-based carrier screening in our study could further clarify the abnormal LC-MS/MS results, which could also discover more monogenic disorders uncovered by LC-MS/MS screening.

A novel 4.9 Kb deletion at beta-globin gene is identified by the third-generation sequencing: Case report from Baoan, China.

BACKGROUND: Thalassemia is a common inherited haemoglobin disorder worldwide, several methods have been utilized in the step-wise screening. Even though hundreds of mutations in globin genes have been reported, novel mutations are continuously emerging as the development of DNA sequencing. METHODS: The case is a 27-year-old female with abnormal values of routine hematological indices, who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays cover 26 mutations in HBA and HBB genes: gap-PCR and agarose gel electrophoresis were performed to detect deletions, while PCR-reverse dot blot was used to detect point mutations. The next- and third- generation sequencing were used to identify the known and potential novel genotypes of thalassemia, and multiplex ligation-dependent probe amplification (MLPA) was used for genotype validation. RESULTS: Hematological results indicate microcytic hypochromic anemia, high HbA2 (7.2%) and high HbF (6.2%). None of the known genotypes of thalassemia were matched for this case, but a novel 4.9 Kb deletion at HBB gene (hg38, Chr11: 5226187-5231089) was discovered by the third-generation sequencing, the novel deletion was also validated by MLPA (8 probes, 11p15.4: 203314-207652). CONCLUSIONS: This study suggests the third-generation sequencing has promising potentiality to discover novel genotypes (especially deletions) of thalassemia.

Low-pass genome sequencing: a validated method in clinical cytogenetics.

Clinically significant copy-number variants (CNVs) known to cause human diseases are routinely detected by chromosomal microarray analysis (CMA). Recently, genome sequencing (GS) has been introduced for CNV analysis; however, sequencing depth (determined by sequencing read-length and read-amount) is a variable parameter across different laboratories. Variating sequencing depths affect the CNV detection resolution and also make it difficult for cross-laboratory referencing or comparison. In this study, by using data from 50 samples with high read-depth GS (30×) and the reported clinically significant CNVs, we first demonstrated the optimal read-amount and the most cost-effective read-length for CNV analysis to be 15 million reads and single-end 50 bp (equivalent to a read-depth of 0.25-fold), respectively. In addition, we showed that CNVs at mosaic levels as low as 30% are readily detected, furthermore, CNVs larger than 2.5 Mb are also detectable at mosaic levels as low as 20%. Herein, by conducting a retrospective back-to-back comparison study of low-pass GS versus routine CMA for 532 prenatal, miscarriage, and postnatal cases, the overall diagnostic yield was 22.4% (119/532) for CMA and 23.1% (123/532) for low-pass GS. Thus, the overall relative improvement of the diagnostic yield by low-pass GS versus CMA was ~ 3.4% (4/119). Identification of cryptic and clinically significant CNVs among prenatal, miscarriage, and postnatal cases demonstrated that CNV detection at higher resolutions is warranted for clinical diagnosis regardless of referral indications. Overall, our study supports low-pass GS as the first-tier genetic test for molecular cytogenetic testing.

Beneficial Effects of Probiotic Treatment on Gut Microbiota in Very Low Birth Weight Infants.

The very low birth weight (VLBW) infant is at great risk for marked dysbiosis of the gut microbiota. In the present study, a total of 36 VLBW infants were randomly divided into two groups, who were treated with combined probiotics and placebo, and 72 fecal specimens on days 14 and 28 of life were collected from them. Finally, 32 fecal specimens extracted from 16 preterm VLBW infants were qualified and analyzed using 16S rRNA gene sequencing. The primary outcome was to evaluate the change of gut microbiota in VLBW infants after combined probiotic supplement. The secondary outcome was to analyze the correlation gut microbial composition and levels of cytokines. We found that probiotic treatment, but not placebo, decreased the α-diversity of gut microbiota in VLBW infants. At the phylum level, probiotic treatment strongly increased the abundance of Firmicutes, whereas that of Proteobacteria was significantly reduced. At the family level, Streptococcaceae and Lactobacillaceae became prevalent after probiotic treatment, while the relative abundance of Enterobacteriaceae was reduced in the meantime. Most notably, significant correlations were observed between Lactobacillaceae abundance and serum cytokine levels. Further studies are required to shed more light on the characteristics of gut microbiota of VLBW neonates. And the modulation of microbiota should be considered to improve the survival rate of VLBW infants.

[A protective effect conferred by KIR3DL1 and its cognate ligand against cervical cancer among ethnic Han Chinese population and its potential mechanism].

OBJECTIVE: To explore the role of inhibitory KIR (iKIR) and its cognate HLA ligand in the occurrence and development of cervical cancer among ethnic Han Chinese and its potential mechanism. METHODS: Peripheral blood samples from 265 Han Chinese patients with cervical intraepithelial neoplasia (CIN)/cervical cancer and 200 ethnically matched healthy controls were collected. The results of KIR PCR-SSP, HLA PCR-rSSO and KIR3DL1 PCR-SBT, together with cervical cancer data from the TCGA database, were used to assess the association of iKIR genes, receptor-ligand gene combinations, iKIR transcription level in the tumor tissue and the KIR3DL1 alleles with the occurrence and development of cervical cancer. RESULTS: Among the four iKIR genes (KIR2DL1, 2DL2/3, 3DL1 and 3DL2), the frequencies of KIR3DL1 and KIR3DL1-HLA-Bw4 genes among controls were significantly higher than those of the cervical cancer group (96.5% vs. 87.0%, P = 0.018; 81.5% vs. 64.8%, P=0.009). The survival rate of cervical cancer patients with a high transcription level of KIR3DL1 in tumor tissues was significantly higher than those with a low/medium transcription level (P=0.028). The frequency of strong-inhibitory and high-expression KIR3DL1*01502 allele among the healthy population was significantly higher than that of the cervical cancer group (76.0% vs. 59.3%, P =0.015). CONCLUSION: Combined KIR3DL1 and KIR3DL1-HLA-Bw4 can confer a protective effect against the development of cervical cancer, which may be attributed to the strong-inhibitory and high-expression allele of KIR3DL1*01502.

p.E95K mutation in Indian hedgehog causing brachydactyly type A1 impairs IHH/Gli1 downstream transcriptional regulation.

BACKGROUND: Brachydactyly type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes. It is the first recorded disorder of the autosomal dominant Mendelian trait. Indian hedgehog (IHH) gene is closely associated with BDA1, which was firstly mapped and identified in Chinese families in 2000. Previous studies have demonstrated that BDA1-related mutant IHH proteins affected interactions with its receptors and impaired IHH signaling. However, how the altered signaling pathway affects downstream transcriptional regulation remains unclear. RESULTS: Based on the mouse C3H10T1/2 cell model for IHH signaling activation, two recombinant human IHH-N proteins, including a wild type protein (WT, amino acid residues 28-202) and a mutant protein (MT, p.E95k), were analyzed. We identified 347, 47 and 4 Gli1 binding sites in the corresponding WT, MT and control group by chromatin immunoprecipitation and the overlapping of these three sets was poor. The putative cis regulated genes in WT group were enriched in sensory perception and G-protein coupled receptor-signaling pathway. On the other hand, putative cis regulated genes were enriched in Runx2-related pathways in MT group. Differentially expressed genes in WT and MT groups indicated that the alteration of mutant IHH signaling involved cell-cell signaling and cellular migration. Cellular assay of migration and proliferation validated that the mutant IHH signaling impaired these two cellular functions. CONCLUSIONS: In this study, we performed integrated genome-wide analyses to characterize differences of IHH/Gli1 downstream regulation between wild type IHH signaling and the E95K mutant signaling. Based on the cell model, our results demonstrated that the E95K mutant signaling altered Gli1-DNA binding pattern, impaired downstream gene expressions, and leaded to weakened cellular proliferation and migration. This study may help to deepen the understanding of pathogenesis of BDA1 and the role of IHH signaling in chondrogenesis.

Co-amplification at lower denaturation temperature-PCR: methodology and applications.

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

Changes in lncRNAs and related genes in ß-thalassemia minor and ß-thalassemia major.

ß-thalassemia is caused by ß-globin gene mutations. However, heterogeneous phenotypes were found in individuals with same genotype, and still undescribed mechanism underlies such variation. We collected blood samples from 30 ß-thalassemia major, 30 ß-thalassemia minor patients, and 30 matched normal controls. Human lncRNA Array v2.0 (8 × 60 K, Arraystar) was used to detect changes in long non-coding RNAs (lncRNAs) and mRNAs in three samples each from ß-thalassemia major, ß-thalassemia minor, and control groups. Compared with normal controls, 1424 and 2045 lncRNAs were up- and downregulated, respectively, in ß-thalassemia major patients, whereas 623 and 349 lncRNAs were up- and downregulated, respectively, in ß-thalassemia minor patients. Compared with ß-thalassemia minor group, 1367 and 2356 lncRNAs were up- and downregulated, respectively, in ß-thalassemia major group. We selected five lncRNAs that displayed altered expressions (DQ583499, X-inactive specific transcript (Xist), lincRNA-TPM1, MRFS16P, and lincRNA-RUNX2-2) and confirmed their expression levels in all samples using real-time polymerase chain reaction. Based on coding-noncoding gene co-expression network and gene ontology biological process analyses, several signaling pathways were associated with three common organ systems exhibiting ß-thalassemia phenotypes: hematologic, skeletal, and hepatic systems. This study implicates that abnormal expression levels of lncRNAs and mRNA in ß-thalassemia cases may be correlated with its various clinical phenotypes.

A novel loss-of-function heterozygous BRCA2 c.8946_8947delAG mutation found in a Chinese woman with family history of breast cancer.

INTRODUCTION: Breast cancer is the most frequent female malignancy worldwide. Among them, some cases have hereditary susceptibility in two leading genes, BRCA1 and BRCA2. Heterozygous germ line mutations in them are related with increased risk of breast, ovarian and other cancer, following autosomal dominant inheritance mode. METHODS AND RESULTS: For purpose of early finding, early diagnosis and early treatment, mutation detecting of BRCA1/2 genes was performed in unselected 300 breast or ovarian patients and unaffected women using next-generation sequencing and then confirmed by Sanger sequencing. A non-previously reported heterozygous mutation c.8946_8947delAG (p.D2983FfsX34) of BRCA2 gene was identified in an unaffected Chinese woman with family history of breast cancer (her breast cancer mother, also carrying this mutation). The BRCA2-truncated protein resulted from the frame shift mutation was found to lose two putative nuclear localization signals and a Rad51-binding motif in the extreme C-terminal region by bioinformatic prediction. And then in vitro experiments showed that nearly all the mutant protein was unable to translocate to the nucleus to perform DNA repair activity. This novel mutant BRCA2 protein is dysfunction. CONCLUSIONS: We classify the mutation into disease causing and conclude that it is the risk factor for breast cancer in this family. So, conducting the same mutation test and providing genetic counseling for this family is practically meaningful and significant. Meanwhile, the identification of this new mutation enriches the Breast Cancer Information Core database, especially in China.

Construction of a disulfide-stabilized diabody against fibroblast growth factor-2 and the inhibition activity in targeting breast cancer.

Fibroblast growth factor-2 (FGF-2) is one of the most important angiogenic factors to promote tumor growth, progression and metastasis. Neutralizing antibodies against FGF-2 may suppress the growth of tumor cells by blocking the FGF-2 signaling pathway. In this study, a disulfide-stabilized diabody (ds-Diabody) that specifically targets FGF-2 was designed. Compared to its parent antibody, the introduction of disulphide bonds in the diabody could significantly increase the stability of ds-Diabody and maintain its antigen binding activity. The ds-Diabody against FGF-2 could effectively inhibit the tube formation and migration of vascular endothelial cells and block the proliferation and invasion of human breast cancer cells. In the mouse model of breast cancer xenograft tumors, the ds-Diabody against FGF-2 could significantly inhibit the growth of tumor cells. Moreover, the densities of microvessels stained with CD31 and lymphatic vessels stained with LYVE1 in tumors showed a significant decrease following treatment with the ds-Diabody against FGF-2. Our data indicated that the ds-Diabody against FGF-2 could inhibit tumor angiogenesis, lymphangiogenesis and tumor growth.

CMKLR1 deficiency influences glucose tolerance and thermogenesis in mice on high fat diet.

Obesity has become a global epidemic disease, contributing to increases in the prevalence of type 2 diabetes. CMKLR1, one of the receptors for chemerin, has a wide range of functions in physiological and pathological activity, including innate and adaptive immunity, inflammation, metabolism and reproduction. In our study, CMKLR1 deficiency did not influence the gain of body weight but did exacerbate glucose intolerance, increase serum insulin level, and promote insulin resistance in mice on high fat diets. The expression of thermogenesis related genes was examined and indicated to decrease in CMKLR1 knockout (KO) mice in both normal and cold environments, which indicated CMKLR1 influence the thermogenesis process. Cold exposure induced significant body mass decrease and improved glucose tolerance and insulin resistance in wild type HFD mice but had no obvious effect on CMKLR1 KO HFD mice. In vitro, loss of CMKLR1 did not significantly influence the differentiation of stromal vascular fibroblasts (SVFs) derived from adipose tissue, but did suppress the expression of thermogenesis related genes. Collectively, these data demonstrate that CMKLR1 deficiency induces inbalance of glucose metabolism and impairs the cold induced-thermogenesis process in high diet models.

Hypermethylated ERG as a cell-free fetal DNA biomarker for non-invasive prenatal testing of Down syndrome.

BACKGROUND: Previous reports have shown that the ERG gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. In this study, we explore the feasibility of hypermethylated ERG as a cell-free fetal (cff) DNA biomarker for non-invasive prenatal testing (NIPT) of Down syndrome. METHODS: We randomly selected 90 healthy pregnant women, including 30 cases at each trimester of pregnancy. In addition, 15 pregnant women were recruited as the case group whose fetuses had been confirmed to have trisomy 21 by amniotic fluid analysis at 18th to 26th week gestation. Using HpaII, MspІ to digest cell-free maternal plasma DNA, we performed SYBR Green PCR to detect methylated sites of ERG sequences, and analyzed the concentrations of cff DNA in maternal plasma in different gestational trimesters and the case group. RESULTS: The ERG median concentrations of the maternal plasma after Hpa II digestion (LG copies/ml) in first, second and third-trimesters were 5.38, 6.10, and 7.04, respectively (Kruskal-Wallis, P<0.01); and that in the trisomy 21 case group was 6.85, which was higher than the second-trimester (Mann-Whitney, P<0.01). CONCLUSIONS: The study demonstrated that ERG gene is hypermethylated in cff DNA but hypomethylated in maternal DNA; and the median concentration of ERG gene in the trisomy 21 case group is higher than that in the gestational trimester matched normal group. ERG gene, as a fetal DNA biomarker, may be useful for NIPT of Down syndrome.

The clinical characteristics and genotype distribution of Chlamydia trachomatis infection in infants less than six months of age hospitalized with pneumonia.

BACKGROUND: Chlamydia trachomatis is a common sexually-transmitted bacterial pathogen. As no routine screening is performed during pregnancy, neonates and infants are at high risk for C. trachomatis infection. The objective of this study was to investigate the morbidity, clinical characteristics and genotype distribution of C. trachomatis pneumonia in infants less than six months of age. METHODS: Clinical manifestations and laboratory results were recorded. Respiratory sputum specimens were tested using RT-PCR targeting C. trachomatis cryptic plasmid. Simultaneously, respiratory virus antigens were detected by direct immunofluorescence and bacterial pathogens were examined by culture in all sputum samples. Positive C. trachomatis samples were further genotyped using a multiplex PCR reverse line blot assay. The relationship between genotype and pneumonia severity was explored. RESULTS: Of 1408 infants, 101 (7.2%) were infected with C. trachomatis. Sixteen of 101 (15.8%) were assessed as severe pneumonia. These severe cases had a higher proportion of viral co-infection (37.5%) compared to mild pneumonia cases (9.4%, P<0.05).Infants with tachypnea (OR 9.2) and wheezing (OR 3.5) were more likely to be classified as severe pneumonia (P<0.05). Amongst 66 C. trachomatis specimens for which a genotyping result was available, seven genotypes were detected, and 39.4% of these specimens contained two or three genotypes. Overall, genotype E (48.5%) was the most frequent, followed by genotype F (42.4%), J (31.8%), D (12.1%), K (10.6%), G (4.5%) and H (3.0%). There were no significant correlations of particular genotypes with severity of disease, although there was a weak indication that more severe pneumonia might be associated with having certain mixed genotypes of C. trachomatis. CONCLUSIONS: The prevalence of C. trachomatis in the population of young hospitalized infants with pneumonia in Shenzhen was very high. The relationship between genotype distribution and severity of pneumonia was not clear based on this study due to small sample size. Further in-depth investigation correlating genotype and disease severity based on a larger population is needed.

Soluble production and function of vascular endothelial growth factor/basic fibroblast growth factor complex peptide.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important proangiogenic factors in tumor procession. The autocrine and paracrine bFGF and the VEGF in tumor tissue can promote tumor angiogenesis, tumor growth, and metastasis. A VEGF/bFGF Complex Peptide (VBP3) was designed on the basis of epitope peptides from both VEGF and bFGF to elicit in vivo production of anti-bFGF and anti-VEGF antibodies. In this study, we reported on the production of recombinant VBP3 using high cell density fermentation. Fed-batch fermentation for recombinant VBP3 production was conducted, and the production procedure was optimized in a 10-L fermentor. The fraction of soluble VBP3 protein obtained reached 78% of total recombinant protein output under fed-batch fermentation. Purified recombinant VBP3 could inhibit tumor cell proliferation in vitro and stimulate C57BL/6 mice to produce high titer anti-VEGF and anti-bFGF antibodies in vivo. A melanoma-grafted mouse model and an immunohistochemistry assay showed that tumor growth and tumor angiogenesis were significantly inhibited in VBP3-vaccinated mice. These results demonstrated that soluble recombinant VBP3 could be produced by large-scale fermentation, and the product, with good immunogenicity, elicited production of high-titer anti-bFGF and anti-VEGF antibodies, which could be used as a therapeutic tumor vaccine to inhibit tumor angiogenesis and tumor growth.

Progress in genotyping of Chlamydia trachomatis.

OBJECTIVE: To review the common genotyping techniques of Chlamydia trachomatis in terms of their principles, characteristics, applications and limitations. DATA SOURCES: Data used in this review were mainly from English literatures of PubMed database. The search terms were "Chlamydia trachomatis" and "genotyping". Meanwhile, data from World Health Organization were also cited. STUDY SELECTION: Original articles and reviews relevant to present review's theme were selected. RESULTS: Different genotyping techniques were applied on different occasions according to their characteristics, especially in epidemiological studies worldwide, which pushed the study of Chlamydia trachomatis forward greatly. In addition, summaries of some epidemiological studies by genotyping were also included in this work for reference and comparison. CONCLUSIONS: A clear understanding of common genotyping techniques could be helpful to genotype C. trachomatis more appropriately and effectively. Furthermore, more studies on the association of genotypes of Chlamydia trachomatis with clinical manifestations should be performed.

Novel mutations c.[5121_5122insAG]+[6859C>T] of the SPG11 gene associated with cerebellum hypometabolism in a Chinese case of hereditary spastic paraplegia with thin corpus callosum.

Hereditary spastic paraplegia (HSP) is a very heterogeneous disease, both genetically and clinically. To date, approximately 52 loci and 31 genes have been reported to be involved in the causality of HSP. The pattern of inheritance of the disease can be autosomal dominant, autosomal recessive, or X-linked recessive. Autosomal recessive HSP with thin corpus callosum (ARHSP-TCC) is one form of this disease, and a recessive gene, SPG11, is responsible for 41-77% of all ARHSP-TCC cases. SPG11 encodes the protein SPATACSIN, which is most prominently expressed in the cerebellum. However, little is known about its function. Despite diverse clinical presentations, diffuse hypometabolism in the cerebellum has not been reported previously. We have identified an HSP-TCC patient that presented with prominent intellectual disability rather than spasticity. (18)Fluorodeoxyglucose positron emission tomography/computed tomography ((18)FDG-PET/CT) examination showed diffuse hypometabolism in both cerebella. Mutation screening of the SPG11 gene using Sanger sequencing identified the novel compound heterozygous mutation c.[5121_5122insAG]+[6859C>T] (p.[I1708RfsX2]+[Q2287X]) in the patient. The mother bears the c.5121_5122insAG mutation, which results in a frameshift and is predicted to truncate the 735 amino acids from the C-terminus, and the father carries the c.6859C>T mutation, which terminates the 157 amino acids from the C-terminus. Therefore, these mutations may result in the loss of function of wild-type SPATACSIN. Our results suggest that SPATACSIN may be involved in cerebella metabolism, and the novel mutations provide more data for the mutational spectrum of this gene, which will aid in the development of quick and accurate genetic diagnostic tools for this disease.

[Effects of hepatitis B virus on human semen parameters and sperm DNA integrity].

OBJECTIVE: To investigate the effects of hepatitis B virus (HBV) in semen on human semen parameters and sperm DNA integrity. METHODS: We detected HBV DNA in the semen samples of 153 HBsAg-seropositive patients by real-time fluorescence quantitative PCR and calculated the sperm nuclear DNA fragmentation index (DFI) by sperm chromatin dispersion (SCD) assay. We compared the semen parameters between the HBV DNA-positive group (A, n = 43) and HBV DNA-negative group (B, n = 110) and analyzed the correlation of sperm DFI with the number of HBV DNA copies in the semen. RESULTS: HBV DNA was detected in 43 (28.1%) of the 153 semen samples. No statistically significant differences were observed in age, semen volume and sperm concentration between groups A and B (P >0.05). Compared with group B, group A showed significantly decreased sperm viability ([58.0 +/- 18.8]% vs [51.4 +/-17.1]%, P<0.05), progressively motile sperm ([29.6 +/- 13.3]% vs [24.5 +/- 10.1]%, P<0.05), average straight-line velocity ([23.7 +/- 4.0] microm/s vs [19.9 +/- 4.5 ] microm/s, P<0.01) and average path velocity ([26.5 +/- 7.0] microm/s vs [23.4 +/- 5.3] microm/s, P<0.01), but remarkably decreased sperm DFI ([19.3 +/- 8.0]% vs [24.2 +/- 9.4]%, P<0.01). The number of HBV DNA copies in semen exhibited a significant positive correlation with sperm DFI (r = 0.819, P < 0.01). CONCLUSION: HBV DNA in semen is not significantly associated with the number of sperm, but may affect sperm viability, velocity and DFI. There is a load-effect relationship between the number of HBV DNA copies in semen and sperm nuclear DNA integrity.