Functional consequences of copy number variants in miscarriage

BACKGROUND: The presence of unique copy number variations (CNVs) in miscarriages suggests that their integral genes have a role in maintaining early pregnancy. In our previous work, we identified 19 unique CNVs in ~40% of studied euploid miscarriages, which were predominantly familial in origin. In our current work, we assessed their relevance to miscarriage by expression analysis of 14 genes integral to CNVs in available miscarriage chorionic villi. As familial CNVs could cause miscarriage due to imprinting effect, we investigated the allelic expression of one of the genes (TIMP2) previously suggested to be maternally expressed in placenta and involved in placental remodelling and embryo development. RESULTS: Six out of fourteen genes had detectable expression in villi and for three genes the RNA and protein expression was altered due to maternal CNVs. These genes were integral to duplication on Xp22.2 (TRAPPC2 and OFD1) or disrupted by a duplication mapping to 17q25.3 (TIMP2). RNA and protein expression was increased for TRAPPC2 and OFD1 and reduced for TIMP2 in carrier miscarriages. The three genes have roles in processes important for pregnancy development such as extracellular matrix homeostasis (TIMP2 and TRAPPC2) and cilia function (OFD1). TIMP2 allelic expression was not affected by the CNV in miscarriages in comparison to control elective terminations. CONCLUSION: We propose that functional studies of CNVs could help determine if and how the miscarriage CNVs affect the expression of integral genes. In case of parental CNVs, assessment of the function of their integral genes in parental reproductive tissues should be also considered in the future, especially if they affect processes relevant for pregnancy development and support. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13039-015-0109-8) contains supplementary material, which is available to authorized users

Gonadal steroids regulate ADAMTS-1 expression in human endometrial stromal cells in vitro

Gonadal steroids are regulators of the ECM remodeling events that occur in the human endometrium during each menstrual cycle. The ADAMTS represent a novel family of MMPs, the best characterized of which is the initially identified member, ADAMTS-1. ADAMTS-1 has recently been found to be spatiotemporally expressed in the human endometrium during the menstrual cycle with mice null-mutant for this ADAMTS subtype also exhibiting endometrial dysfunction. To date, the factors capable regulating ADAMTS-1 in the human endometrium have not been identified. In view of these observations, I hypothesized that ADAMTS-1 plays a central role in the steroid-mediated remodeling events that occur in the human endometrium during each reproductive cycle. In the studies presented in this thesis, I have examined the ability of the gonadal steroids, progesterone (P4), 17β-estradiol (E2) or the non-aromatisable androgen, dihydrotestosterone (DHT), alone or in combination to regulate ADAMTS-1 mRNA and protein levels in primary cultures of human endometrial stromal cells in a time- and concentration-dependent manner. In addition, I determined whether the anti-steroidal compounds, RU486 (an antiprogestin), ICI 182, 780 (an anti-estrogen) or hydroxflutamide (an anti-androgen) were capable of inhibiting the regulatory effects of these gonadal steroids on stromal ADAMTS-1 levels. Real-time PCR and Western blotting revealed that P4 and DHT increased ADAMTS-1 expression levels whereas E2 alone had no regulatory effect on the expression levels of this ADAMTS subtype in these primary cell cultures. A combination of DHT and P4 potentiated the increase in the levels of the ADAMTS-1 protein species present in these cell cultures whereas E2 was capable of attenuating the stimulatory effects of both P4 and DHT on stromal ADAMTS-1 mRNA and protein expression levels. In contrast, RU486 and hydroxyflutamide specifically inhibited the increase in ADAMTS-1 expression levels mediated by P4 and DHT, respectively. In summary my studies, demonstrate that the regulation of ADAMTS-1 mRNA and protein expression levels in human endometrial stromal cells by gonadal steroids involves a complex interplay between progestins, estrogens and androgens.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

Regulated expression of aggrecanases of ADAMTS family in endometrial physiology and pathology

The ADAMTS (A Disintegrin and Metalloproteinase with TromboSpondin Repeats) are a novel family of secreted metalloproteinases. There is increasing evidence that distinct ADAMTS subtypes play key roles in embryonic development, reproduction and cancer. Nineteen ADAMTS subtypes have been identified in humans but most of them have been characterized only at the structural level. ADAMTS-1, -4, -5, -8, -9 and -15 have been subclassified into a subfamily, known as aggrecanases, owing to their ability to cleave the important extracellular matrix components, versican and aggrecan. Previous studies have determined that ADAMTS-1 and -5 are expressed in first trimester decidual cells and are regulated by IL-1ß and TGF- ß1. I have now found that gonadal steroids have complex regulatory effects upon ADAMTS-1 mRNA and ADAMTS-1 levels in endometrial stromal cells during the human menstrual cycle. I further demonstrate that progesterone (P4) and 5α-dihydrotestosterone (DHT), differentially regulated ADAMTS-5, -8, and -9 mRNA and protein levels in human endometrial stromal cells, suggesting that aggrecanases contribute to steroid-mediated ECM remodeling in the endometrium in preparation for pregnancy. My loss- and gain- of function studies have confirmed a function for ADAMTS-1 in endometrial cancer invasion. Overexpression of ADAMTS-1 in well-differentiated ECC-1 endometrial carcinoma cells promoted cell invasion. In contrast, siRNA-mediated silencing of endogenous ADAMTS-1 in poorly differentiated KLE cells decreased their invasive capacity. I have also found that 17ß-estradiol (E2) can up-regulate ADAMTS-1 mRNA and protein levels in ECC-1 cells. This suggests that ADAMTS-1 plays an important role in endometrial cancer progression, and that E2 promotes well-differentiated endometrial cancer cell invasion, at least in part by specific up-regulating ADAMTS-1 expression. Overall, my research provides useful insight into the molecular mechanisms that regulate endometrial physiology and pathology, and additional support for the concept that ADAMTS represent potentially useful prognostic biomarkers of recurrent pregnancy loss and endometrial cancer.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

Inverted duplication, triplication and quintuplication through sequential breakage‐fusion‐bridge events induced by a terminal deletion at 5p in a case of spontaneous abortion

Abstract Background Integrated chromosome, fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) analyses have been effective in defining unbalanced chromosomal rearrangements. Discordant chromosome and aCGH results are rarely reported. Methods Routine cytogenomic analyses and literature review were performed in the study of a case from products of conception (POC). Results Chromosome and FISH analysis revealed a mosaic pattern consisting of a primary aberration of an inverted duplication of 5p and derived secondary and tertiary aberrations from sequential triplication and quintuplication of 5p, respectively. The aCGH analysis detected only a 1.521 Mb terminal deletion at 5p15.33 with no other pathogenic copy number variants in the genome. This mosaic karyotypic pattern likely resulted from chromosome instability induced by sequential breakage‐fusion‐bridge events during in vitro cell culture. A review of literature found heterogeneous distal deletion and inverted duplication of 5p in prenatal and pediatric cases. Conclusion This is the first case reported in POC with a unique mosaic pattern and discordant chromosome and aCGH results. Caution should be applied in reporting and interpreting these discordant results and further analysis for underlying mechanism should be considered

Integrated FISH, Karyotyping and aCGH Analyses for Effective Prenatal Diagnosis of Common Aneuploidies and Other Cytogenomic Abnormalities

Current prenatal genetic evaluation showed a significantly increase in non-invasive screening and the reduction of invasive diagnostic procedures. To evaluate the diagnostic efficacy on detecting common aneuploidies, structural chromosomal rearrangements, and pathogenic copy number variants (pCNV), we performed a retrospective analysis on a case series initially analyzed by aneuvysion fluorescence in situ hybridization (FISH) and karyotyping then followed by array comparative genomic hybridization (aCGH). Of the 386 cases retrieved from the past decade, common aneuploidies were detected in 137 cases (35.5%), other chromosomal structural rearrangements were detected in four cases (1%), and pCNV were detected in five cases (1.3%). The relative frequencies for common aneuploidies suggested an under detection of sex chromosome aneuploidies. Approximately 9.5% of cases with common aneuploidies showed a mosaic pattern. Inconsistent results between FISH and karyotyping were noted in cases with pseudo-mosaicism introduced by culture artifact or variable cellular proliferation from cells with mosaic karyotypic complements under in vitro cell culture. Based on findings from this case series, cell-based FISH and karyotyping should be performed to detect common aneuploidies, structural chromosomal abnormalities, and mosaic pattern. DNA-based aCGH and reflex FISH should be performed to detect and confirm genomic imbalances and pCNV. Practice points to ensure the diagnostic accuracy and efficacy were summarized

Geographical Variation Reveals Strong Genetic Differentiation in <i>Cryptomeria japonica</i> var. <i>sinensis</i>

The adaptive capacity of tree species is crucial for their survival under environmental change. Liushan (Cryptomeria japonica var. sinensis), an allogamous conifer species, is widely distributed across southern China. However, despite its broad distribution, there have been few investigations on the geographical variation and environmental adaptability of this species. Here, we combined the phenotypic (eight needle traits) and genetic data (14 nSSR loci) to fill this gap by assessing the genetic variation of geographical populations and exploring environmental adaptations of this species. Both phenotypic and molecular genetic analyses indicated a strong genetic differentiation among geographic populations. All populations could be clustered into three groups that were consistent with their geography. Most of the needle traits showed significantly correlated with geography and environmental factors. Geographical isolation and environmental differences are the main factors that have shaped current morphological traits and patterns of genetic variation. We suggest conservation measures to be implemented on a population level with existing populations, especially those with rare phenotypes as the main goal. Our findings shed light on the geographic variation in Liushan and expanded the knowledge of its putative adaptive mechanisms, ultimately benefiting the conservation of this species

High expression of HSP60 and survivin predicts poor prognosis for oral squamous cell carcinoma patients

Abstract Background HSP60 is a heat shock proteins (HSPs) family member and help mitochondrial protein to fold correctly. Survivin is one of the inhibitors of apoptosis protein family member, which plays a significant part in cancer progression. They were capable of forming HSP60-survivin complexes and involved in the development of various tumors. Methods The Cancer Genome Atlas (TCGA) database demonstrated that HSP60 and survivin and their correlation on mRNA expression level with OSCC patients. Besides, expression of HSP60 and survivin proteins was studied utilizing immunohistochemistry in tissue microarrays (TMA) in OSCC and in adjacent non-cancerous squamous epithelium (Non-CCSE) tissues. Results Significantly increased levels of HSP60 and survivin in most cancers compared to normal tissue by pan-cancer analysis. HSP60 and survivin proved a significantly increased expression in OSCC samples compared to Non-CCSE both on mRNA and protein (both P < 0.05). Additionally, elevated HSP60 displayed a positive correlation with survivin in terms of mRNA and protein expression levels (all P < 0.001). Patients with OSCC who had advanced clinical stage or lymph node metastasis (LNM) showed higher HSP60 expression (P = 0.004, P = 0.006, respectively). Higher levels of the proteins HSP60 and survivin were significantly inversely correlated relationship with OSCC patients’ overall survival rates in multivariate survival analysis (P = 0.018, P = 0.040). From the above results, overexpression of HSP60 and survivin protein may serve as independent biomarkers predicting poor prognosis in OSCC. Conclusions Elevated HSP60 and survivin might be served as novel poor prognosis biomarkers for surgically resected OSCC patients

Estimation on risk of spontaneous abortions by genomic disorders from a meta‐analysis of microarray results on large case series of pregnancy losses

Abstract A meta‐analysis on seven large case series (>1000 cases) of chromosome microarray analysis (CMA) on products of conceptions (POC) evaluated the diagnostic yields of genomic disorders and syndromic pathogenic copy number variants (pCNVs) from a collection of 35,130 POC cases. CMA detected chromosomal abnormalities and pCNVs in approximately 50% and 2.5% of cases, respectively. The genomic disorders and syndromic pCNVs accounted for 31% of the detected pCNVs, and their incidences in POC ranged from 1/750 to 1/12,000. The newborn incidences of these genomic disorders and syndromic pCNVs were estimated in a range of 1/4000 to 1/50,000 live births from population genetic studies and diagnostic yields of a large case series of 32,587 pediatric patients. The risk of spontaneous abortion (SAB) for DiGeorge syndrome (DGS), Wolf–Hirschhorn syndrome (WHS), and William–Beuren syndrome (WBS) was 42%, 33%, and 21%, respectively. The estimated overall risk of SAB for major genomic disorders and syndromic pCNVs was approximately 38%, which was significantly lower than the 94% overall risk of SAB for chromosomal abnormalities. Further classification on levels of risk of SAB to high (>75%), intermediate (51%–75%), and low (26%–50%) for known chromosomal abnormalities, genomic disorders, and syndromic pCNVs could provide evidence‐based interpretation in prenatal diagnosis and genetic counseling

Analytical validation and chromosomal distribution of regions of homozygosity by oligonucleotide array comparative genomic hybridization from normal prenatal and postnatal case series

Abstract Background Regions of homozygosity (ROH) are continuous homozygous segments commonly seen in the human genome. The integration of single nucleotide polymorphism (SNP) probes into current array comparative genomic hybridization (aCGH) analysis has enabled the detection of the ROH. However, for detecting and reporting biologically relevant ROH in a clinical setting, it is necessary to assess the analytical validity of SNP calling and the chromosomal distribution of ROH in normal populations. Methods The analytical validity was evaluated by correlating the consistency of SNP calling with the quality parameters of aCGH and by accessing the accuracy of SNP calling using PCR based restriction enzyme digestion and Sanger sequencing. The distribution of ROH was evaluated by the numbers, sizes, locations, and frequencies of ROH from the collection of data from parental, postnatal, and prenatal case series that had normal aCGH and chromosome results. Results The SNP calling failure rate was 20–30% with a derivative Log2 ratio (DLR) below 0.2 and increased significantly to 30–40% with DLR of 0.2–0.4. The accuracy of SNP calling is 93%. Of the 958 cases tested, 34% had no ROH, 64% had one to four ROH, and less than 1% had more than five ROH. Of the 1196 ROH detected, 95% were less than 10 Mb. The distribution of numbers and sizes of ROH showed no differences among the parental, pediatric and prenatal case series and test tissues. The chromosomal distribution of ROH was non-random with ROH seen most frequently in chromosome 8, less frequently in chromosomes 2, 6, 10, 12, 11 and 18, and most rarely seen on chromosomes 15, 19, 21 and 22. Recurrent ROH occurring with a frequency greater than 1% were detected in 17 chromosomal loci which locates either in the pericentric or interstitial regions. Conclusion With a quality control parameter of DLR set at below 0.2, the consistency of SNP calling would be 75%, the accuracy of SNP call could be 93%, and the observed chromosomal distribution of ROH could be used as a reference. This aCGH analysis could be a reliable screening tool to document biologically relevant ROH and recommend further molecular analysis

Novel [1,2,3]triazolo[4,5-d]pyrimidine derivatives containing hydrazone fragment as potent and selective anticancer agents

In this paper, based on molecular hybridization, a series of [1,2,3]triazolo[4,5-d]pyrimidine derivatives containing hydrazine was synthesized and their antiproliferative activities against 5 cancer cell lines (MGC-803, PC3, PC9, EC9706 and SMMC-7721) were evaluated. We found that most of them exhibited obvious growth inhibition effects on these tested cancer cells, especially compound 34 on PC3 cells (IC50 = 26.25 ± 0.28 nM). Meanwhile, compound 34 displayed best selectivity on PC3, compared with the other cancer cell lines, as well as excellent selectivity towards normal cell lines (Het-1A, L02 and GES-1). Further investigations demonstrated that 34 could significantly inhibit PC3 cells’ colony formation, increase cellular ROS content, suppress EGFR expression and induce apoptosis. Our findings indicate that 34 may serve as a novel lead compound for the discovery of more triazolopyrimidine derivatives with improved anticancer potency and selectivity