Quantitative fluorescence PCR for rapid prenatal diagnosis of common chromosome aneuploidies

AIM:To evaluate the effectiveness of quantitative fluorescence PCR(QF-PCR) as a method for rapid prenatal diagnosis of common chromosome aneuploidyies. METHODS:Polymorphism information contents(PIC) of each short tandem repeat(STR) in 95 cases of amniotic fluid samples were detected by QF-PCR and the data were compared with the results of routine chromosome karyotype analysis. RESULTS:The results of QF-PCR included 63 normal samples and 14 trisomy 21, 4 trisomy 18, 3(45, X) and 8(47, XXX). The rapid QF-PCR assay was successful to detect all aneuploidies involving chromosomes 21, 18, 13, X and Y in prenatal diagnosis, which were verified by chromosome karyotype analysis. The results showed that the total coincidence rate between QF-PCR and routine chromosome karyotype analysis was 96.8%. CONCLUSION:The QF-PCR is a reliable method of detecting common chromosome aneuploidies for rapid prenatal diagnosis.     

Value of single nucleotide polymorphism array in investigating origin and pathogenic mechanisms of uniparental disomy

AIM：To assess the value of the genome-wide human single nucleotide polymorphism (SNP) array for the investigation of the origin and pathogenic mechanisms and the genetic counseling of uniparental disomy (UPD). METHODS：Genetic analysis with the genome-wide human SNP array was carried out on the fetal cells in amniotic fluid and the peripheral blood cells from 124 pregnant women with high risk of Down’s syndrome, whose G-banded chromosome karyotype analysis was done using the fetal cells in amniotic fluid. The peripheral blood cells from the fetuses fathers were also taken for SNP array analysis. RESULTS：Two cases of segmental UPD 16 were found from the SNP array analysis of the fetal cells in amniotic fluid. The regions of isodisomy in one case were located in 16p12.2~13.3 and 16q24.1~24.3, and the region of the other was located in 16q21~24.3. Both cases of UPD were maternal upon the genetic linkage analysis of the peripheral blood cells of the parents. CONCLUSION：The genes that induce the fetal growth restriction are probably located at the ends of long arm and short arm of chromosome 16. SNP array can identify the parental origins and the pathogenic mechanisms of UPD, which provides the assistance for genetic counseling.     

Noninvasive prenatal diagnosis of β-thalassemia by enriching cell-free fetal DNA in materal plasma

AIM: To establish a kind of simple and efficient method for cell-free fetal DNA (cff-DNA) enrichment and to investigate its range of applications and the advantages and disadvantages.METHODS: (1) The single nucleotide polymorphisms(SNPs), which linked to paternal β-thalassemia mutations, were screened. We analyzed the contact between the SNPs in β- thalassemia gene (HBBgene) and haploid type by the Haploview software, and then selected these close SNPs which have higher heterozygosity with the HBBgene. (2) We selected 4 cases of different β-thalassemia mutations with their husband, and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma. If the IVS-II-654 mutation was not detected, we detected the SNP which linked to the IVS-II-654 mutation. Similarly, we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma. At the same time, we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma.RESULTS: (1) Nine cases of the SNP (rs7480526) linked to the mutation at IVS-II-654 in HBBgene, and 11 cases of the SNP (rs10768683) linked to the mutation at CD41-42 in HBBgene were detected. (2) We detected 1 case who inherited the paternal β-thalassemia mutation (IVS-II-654). We did not directly detect patermal IVS-II-654 mutation in maternal plasma, but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100% detection, and 2 cases inherited the paternal β-thalassemia mutations (CD41-42) in the maternal plasma by TT-FULL-COLD-PCR and had 100% detection. However, we detected nothing by conventional PCR. CONCLUSION: TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.     

Diagnostic validity of cervical precancerous lesions by liquid-based cytology technique combined with high-risk HPV-DNA detection

AIM: To investigate liquid-based cytology technique (LCT) combined with high-risk human papilloma virus DNA (HPV-DNA) detection for diagnosis of cervical precancerous lesions. METHODS: Screening of cervical cancer was performed by LCT combined with HPV-DNA detection in 6 521 women at the age of 19~65 in our hospital for physical examination from December 2007 to December 2010. Eighteen high-risk HPV isoform genes were detected by HPV typing gene chip detection system. The women with positive detection underwent colposcopic cervical biopsies. The women with negative detection also underwent colposcopic cervical biopsies if the operation was of their own accord. RESULTS: The LCT positive results (≥ASCUS) were observed in 152 cases and HPV positive results were obtained in 86 cases. The women with both 2 positive results were found in 42 cases. The women with both LCT and HPV negative results were determined in 6 325 cases. In 152 cases of LCT positive and 86 cases of HPV positive women, the positive results of pathological biopsy (≥CINI) were 112 and 68 cases, respectively. Thirty-four cases in the 42 cases of both LCT and HPV positive women were conformed by pathological biopsy. In 6 325 women with negative results of LCT and HPV detection, 2 000 were perform pathological biopsy according to their own accord and only 1 had positive result. The diagnostic sensitivity of LCT for cervical precancerous lesions was 76.19% with the specificity of 98.05%. The diagnostic sensitivity of HPV detection for precancerous lesions of uterine cervix was 46.26% with the specificity of 99.12%. Combination of the 2 methods to diagnose cervical precancerous lesions made the sensitivity increase to 99.32% with the specificity of 99.61%. CONCLUSION: Liquid-based cytology technique combined with high-risk HPV-DNA detection is better than each of the single technique for screening precancerous lesion of cervical carcinoma.     

Characteristics of chromosome of teratocarcinoma and its influence elements

AIM:To investigate the characteristics of chromosome of teratocarcinoma and its influence elements.METHODS:We used the methods of G-banded karyotype analysis,DNA basic sequence analysis and Western blot and the others,and studied the chromosome karyotype and the status of p53 gene of teratocarcinoma PA-1 cell line which had been cultured for 407-445 passages for 20 years. RESULTS: More than 80% PA-1 cells still maintained the near-diploid karyotype,after passage 30 the cells appeared with M1 and M2 chromosomal markers because of a balanced translocation between chromosomes 15 and 20. DNA directional sequence analysis of RP-PCR products revealed that there were both wild and mutated band (p53 codon 239 mutation), Western blot did not detect mutational p53 gene protein,while p21 protein expression lower than that in normal human fibroblasts. CONCLUSION:Missense mutation of one of the p53 allele gene of PA-1 cells in human teratocarcinoma was detected after cultured for more than twenty years, which was not sufficient to induce the instability of the chromosome of cell line.     

葡萄果粒形状简化基因组关联分析

为了寻找控制葡萄果粒形状的基因位点,本研究中对304个葡萄种质的果形性状了进行简化基因组关联分析。对葡萄种质材料的基因组DNA进行测序,平均测序深度为8.13×,共开发466 618个SLAF标签,其中多态性SLAF标签有392 374个。比对测序分析获得了4 241 729个SNP标记,进一步根据完整度大于0.8以及次要基因型频率大于0.05的标准进行过滤,共得到481 192个SNP。本次检测到的控制葡萄果粒形状的相关位点为首次报道,除基于一般线性模型的SNP rs26137624外,其余检测表型遗传变异贡献率均大于11%。基于一般线性模型和混合线性模型分别关联到7个和3个显著性SNP标记,且均位于2号染色体上。通过筛选每个显著性SNP标记上下游区域,对关联区域进行功能基因挖掘,共得到10个有预测功能注释的基因和7个未知功能的基因。     

Methylation markers in placenta tissues of euploid and trisomy 21

AIM:To explore the DNA methylation markers for non-invasive prenatal diagnosis of trisomy 21. METHODS:The DNA methylation ratios of 3 genes (4 fragments) on chromosome 21 (CGI149, CGI045, HLCS-1 and HLCS-2) in blood cells from 13 non-pregnant women, and in placental tissues from 15 euploid and 11 trisomy 21 pregnant women were measured using the methods of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and bisulfite sequencing in combination. RESULTS:The results obtained from MS-MLPA were consistent with the results from bisulfite sequencing. The fragments of CGI149, CGI045 and HLCS-2 were unmethylated in the non-pregnant woman blood cells. HLCS-1 and HLCS-2 were methylated in all euploid and all trisomy 21 placentae. CGI149 was methylated in 13 (13/15) of the euploid and 10 (10/11) of the trisomy 21 placental tissues. CGI045 was methylated in 11 (11/15) of the euploid and all the Trisomy 21 placental tissues. Only HLCS-2 was found to be methylated in all placental tissues but unmethylated in all non-pregnant woman blood cells. Only the DNA methylation ratio in CGI149 was significantly different between euploid and trisomy 21 placental tissue (P<0.05). No difference among HLCS-1, HLCS-2 and CGI045 was observed. CONCLUSION: MS-MLPA is an effective alternative to bisulfite sequencing for the assessment of methylation ratios in placental tissue. CGI149, CGI045, HLCS-1 and HLCS-2 are not appropriate DNA methylation markers for non-invasive prenatal diagnosis of trisomy 21.     

黄瓜叶色黄化突变基因yl-2的鉴定

叶绿素的含量与叶片光合作用效率以及作物产量潜力密切相关,是作物的一个重要生理指标。通过对黄瓜株系HN3种子进行甲磺酸乙酯(EMS)诱变,筛选得到叶色黄化的突变体yl-2。以该叶色黄化突变体yl-2和野生型黄瓜HN3为亲本,构建了BC_1F_2分离群体,遗传分析表明该突变体为隐性单基因控制。通过DNA混池测序,运用MutMap的方法寻找突变基因,得到4个符合要求的SNP位点,这4个SNP位点分布在黄瓜6号染色体上13～19 Mb的物理区间内。在这4个SNP中,仅有1个SNP即SNP17451330位于基因(Csa6G385090)上,且为异义突变,其余3个SNP均位于基因间。生物信息分析发现基因Csa6G385090编码叶绿素a加氧酶CAO,SNP17451330导致的氨基酸突变位于CAO蛋白的保守结构域。根据异义突变SNP17451330设计的dCAPS标记与群体植株的叶色表型共分离,预示着基因Csa6G385090很可能是叶色黄化突变体yl-2的候选基因。     

Evaluation of antisperm antibody in infertile women with polycystic ovary syndrome

AIM: To investigate the level of circulatory antisperm antibody (ASA) in infertile women with polycystic ovary syndrome (PCOS). METHODS: A total of 127 infertile women with PCOS (26~35 years old) were divided into 2 groups according to the body mass index (BMI):obese group (BMI ≥ 25 kg/m², n=42) and non-obese group (18 kg/m² < BMI < 25 kg/m², n=85). The infertile women aged 24~36 years of normal weight without PCOS (n=90) were chosen as controls (control group; 18 kg/m² < BMI < 25 kg/m²). The serum levels of ASA-IgG and ASA-IgA were screened by the indirect immunobead test according to the WHO laboratory manual. The 50% or more of the motile sperm attached to one or more immunobeads were regarded as clinical positivity according to the WHO criteria. The 20%~49% motile sperm that had adherent particles were deemed to be weakly positive. Less than 20% was negative. RESULTS: No case in obese group and non-obese group showed the positive level of ASA-IgG. Two cases in control group were detected to be ASA-IgG positive (2.2%). One case in obese group (2.4%), 2 cases in non-obese group (2.2%) and 2 cases (2.2%) in control group were detected to be ASA-IgG weakly positive, and no difference was found in the weakly-positive incidence among these 3 groups. ASA-IgA was not detected in all the cases. CONCLUSION: The circulatory ASA is not associated with the pathogenesis of infertile women with PCOS. The detection of ASA still needs to be routinely performed for infertile women.     

17种绣线菊核型特征及核型参数分析

以17种(品种)绣线菊为材料,利用改良去壁低渗法分析染色体特征并作相关性和聚类分析,探讨核型特征与种间进化关系。研究结果表明:供试绣线菊染色体数稳定,为2n=18和2n=36,染色体基数为9,主要由中部着丝点染色体(m)和近中部着丝点染色体(sm)组成。绒毛绣线菊、石蚕叶绣线菊、楔叶绣线菊染色体中有随体结构;核型类型为1A、1B、2B,染色体平均臂比范围为1.26～1.64,最长与最短染色体比值范围为1.67～3.51,着丝点指数范围为38.96%～44.26%,核型不对称系数范围为56.14%～60.69%;按核型不对称系数从高到低排序其进化程度,石棒绣线菊、绒毛绣线菊和楔叶绣线菊等进化程度高,珍珠绣线菊、石蚕叶绣线菊和三亚绣线菊等进化程度低;核型参数的重要性排序为核型不对称系数平均臂比臂比大于2的比率染色体最长/最短值核型类型着丝点指数均值;17种绣线菊核型参数在种间存在差异,可作为绣线菊属植物分类依据;核型特征聚类分析将17种绣线菊分为4组。     

Karyotype study on 15 populations of Eremostachys laciniata Bunge in Iran

A karyotype study was performed on 15 populations of Eremostachys laciniata Bunge from Iran in order to quantify the extent of cytological variation for use in breeding programmes. Ten mitotic root tip cells at the metaphase stage were prepared from each population by the squash method. The chromosomes of suitable mitotic cells were counted and various parameters, including long and short arm lengths, chromosome lengths, total length of the haploid chromosome complement, arm ratios, and the centromeric index, were measured. All 15 populations were diploid with 2n = 2x = 22 and were differentiated by their karyotypic parameters. All 15 E. laciniata populations occupied Class 1A of Stebbins’ karyotype classification, indicating the presence of a primitive symmetrical karyotype. The size of the mitotic chromosomes was medium, and mean chromosome lengths ranged from 3.84 to 4.77 μm. The shortest chromosome lengths were observed in the Ajabshir population and the longest in the Heydarabad population. The Heydarabad population also had the longest total chromosome length and the Ajabshir population had the shortest total chromosome length. The centromeric indices of the chromosome complements varied from 39.3% to 43.7%. Metacentric chromosomes were the most common in all populations, whereas submetacentric chromosomes were rare. The A₁ index varied from 0.215 (Areshtanab) to 0.337 (Til). The A₂ index ranged from 0.116 (Til) to 0.143 (Kaleybar), and the karyotype asymmetry index varied between 0.051 and 0.097. The results of a principal component analysis (PCA) showed that the main elements of the first three main principal components accounted for 97% of the total variance. Cluster analysis by Ward’s method indicated that all 15 populations could be classified into three clusters. Finally, the results of the karyotypic parameters showed that the Til, Heydarabad, and Marand populations had higher levels of karyotypic heterogeneity and may be useful when developing breeding programmes.     

Osteogenic and neurogenic differentiation of human yolk sac mesenchymal stem cells

AIM: To purify human yolk sac mesenchymal stem cells (hYS-MSC) and investigate its osteogenic and neurogenic differentiation potentials. METHODS: hYS-MSC were separated from yolk sac and purified via passage culture. The karyotype of hYS-MSCs was analyzed via G-banded characteristics. Flow cytometric analysis was used to determine the cell cycle and phenotype of hYS-MSC. The AKP expression of hYS-MSC was also tested. Osteogenic differentiation of hYS-MSCs was induced by 10-8mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 mg/L vitamin C. Alizarin red S stain was used for identification of mineralization. β-mecaptoethanol or salviae miltiorrhizae were used to induce neurogenic differentiation of hYS-MSCs. The expressions of NSE, NF and GFAP were identified by immunohistochemical method. RESULTS: hYS-MSCs could be purified at passages 2 or 3. The cell cycle analysis suggested that hYS-MSCs showed strong proliferational potentials by which the cells kept normal diploid karyotype during the in vitro culture. Flow cytometry showed the phenotype of purified hYS-MSCs was uniformly positive for CD29, CD44, CD105, and CD166, and negative for reactivity to antigens CD34, CD45, or CD86. hYS-MSCs were weakly but clearly positive in AKP. Osteogenic differentiation was appeared after induction of osteogenic differentiation. hYS-MSCs, which were of spindle shape, uniform in size, were induced to pleomorphism osteoblast-like cells which expressed high level of AKP. Aggregates or nodules were formed at day 7 and calcium accumulation was detected by alizarin red S staining on day 10 or day 14. Neurogenic differentiation of hYS-MSCs was induced by β-mecaptoethanol or salviae miltiorrhizae. NSE, NF or GFAP positive cells were detected by immunohistochemical staining. CONCLUSIONS: hYS-MSCs have strong proliferation potential and the normal diploid karyotype is kept during the in vitro culture. The phenotype of hYS-MSCs is coincident with adult hMSCs. hYS-MSCs could be induced to differentiate into osteogenic or neurogenic cells.     

叶籽银杏种质资源染色体核型分析及进化趋势

 以来自日本、中国山东等地的28株叶籽银杏种质幼叶为试材,采用常规压片法,对其核型进行了研究。结果表明：各种质染色体均为二倍体,2n = 2x = 24,核型有中部着丝粒染色体（m）、近中部着丝粒染色体（sm）和近端部着丝粒染色体（st）等3种类型;有6个种质具随体。方差分析表明：种质SY2染色体长度比与其它种质差异显著,为3.97;各种质的平均臂比和核型不对称系数之间差异较大。种质GX3和种质HB较进化,种质NS2较原始。     

应用生物学软件分析四季橘染色体核型和带型

【目的】为了改进以往人工方式观测染色体精准度的不足,【方法】采用MicroMeasure软件和Image-Pro Plus图像分析软件对4,6-联脒-2-苯基吲哚(DAPI)荧光染色的四季橘(Citrus madurensis Lour.Tseng)体细胞中期染色体分别进行核型分析和三维重建。【结果】得到四季橘的核型公式2n=18=16 m(2SAT)+2 sm,其核型类型为1B型,属于进化中较原始类型;获得四季橘染色体立体图,并依据每条前中期染色体(从短臂到长臂)DAPI荧光强度变化,构建出染色体带型模式图。【结论】该研究利用染色体图像分析软件对染色体进行识别,获得了较丰富的染色体核型数据信息,为柑橘细胞遗传学研究提供了新方法。     

百合杂交育种中染色体研究进展

 综述了染色体的研究方法及其在百合属植物中的应用,包括核型分析、Giemsa C–带技术、原位杂交技术。对百合倍性杂交的结果,多倍体育种的方式以及不同倍性百合作为亲本进行杂交的规律也进行了讨论。     

薹菜的核型分析

采用根尖压片法对薹菜进行染色体数目和核型分析。结果表明：薹菜为二倍体,染色体数2n=20,核型公式为K（2n）=2X=20=7M+2T+St,染色体相对长度组成为2n=20=2L+4M2+4S,染色体组型为2A型。     

Single nucleotide polymorphism and mutation of miRNA binding sites at BCL11B-3'UTR in healthy individuals and patients with T-ALL

AIM:To analyze the microRNA (miRNA) binding sites at B-cell lymphoma/leukemia 11B gene 3'-untranslated region (BCL11B-3'UTR), and to establish the method of identifying the single nucleotide polymorphism (SNP) and mutation of these miRNA binding sites in healthy individuals and patients with T-cell acute lymphoid leukemia (T-ALL). METHODS:TargetScan was used for screening and predicting the miRNA binding sites at BCL11B-3'UTR. PCR and sequencing were used to identify the miRNA binding sites at BCL11B-3'UTR. The polymorphisms in DNA sample of peripheral blood mononuclear cells from 20 healthy individuals and 21 patients with T-ALL at this region were analyzed. RESULTS:Twenty-four highly conserved miRNA binding sites were screened according to the criteria of context ++ score and seed match categories. The nucleotide exchange (T>C) located at site 2 402 of BCL11B-3'UTR was detected in one case out of 21 cases of T-ALL samples, which had been registered as SNP (rs184678181) in dbSNP. No polymorphism or mutation in BCL11B-3'UTR miRNA binding sites was identified in the samples from the healthy individuals. CONCLUSION:Polymorphism or mutation in BCL11B-3'UTR is rare in healthy individuals and T-ALL patients. To our best knowledge, it is the first identification of BCL11B-3'UTR SNP (T>C at site 2 402), which is involved in hsa-miR-6814-5p binding site in a patient with T-ALL. Further investigation will focus on its effect for BCL11B expression regulation.     

Correlation of adiponectin gene SNP+45 T/G polymorphism with coronary heart disease

AIM: To investigate the relationship between adiponectin gene SNP+45 polymorphism and coronary heart disease (CHD) in south China Han population. METHODS: The nondiabetic CHD patients diagnosed by the coronary angiography were selected as CHD subjects (153 cases), and 73 healthy adults served as normal control subjects. The polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) was performed to identify the distribution pattern of adiponectin gene SNP+45 in all subjects. The levels of plasma adiponectin were measured by ELISA. RESULTS: The frequency of T/G + G/G genotype and G allele in CHD patients were significantly higher than those in control subjects (P<0.05). Logistic regression analysis revealed that the adiponectin gene SNP+45 T/G+G/G genotype had a strong positive association with CHD (OR: 2.132, 95.0% CI: 1.034-4.397, P＜0.05). The plasma adiponectin was negatively associated with CHD (OR: 0.868, 95.0% CI: 0.785-0.959, P<0.05). CONCLUSION: The T/G+ G/G genotype was a possible risk factor for CHD in southern China Han population.     

二乔刺槐染色体核型分析及有丝分裂过程观察

利用茎尖压片法对二乔刺槐染色体的数目进行了统计,并进行了核型分析,观察了细胞有丝分裂过程.结果表明:二乔刺槐单倍体染色体数为2n=22,核型公式为2n=2x=22=4m+10sm+2st+6t;属于"3B"型;核型不对称系数为72.14%.二乔刺槐有丝分裂前期染色体逐渐浓缩变粗,中期染色体清晰可见,后期染色单体发生分离并向相反的两极移动,末期形成新的子核.