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Multiple molecular tests are currently needed for accurate carrier testing for thalassemia. Therefore, long-molecule sequencing (LMS) was evaluated as an alternate on the PacBio Sequel platform for genotyping carriers of α-thalassemia or β-thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and β (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using FreeBayes software version 1.2.0. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common South East Asian, -α3.7, and -α4.2 deletions and four different rare single-nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the most common Chinese insertion and deletion variant c.126_129delCTTT and 14 different SNVs/insertions and deletions and could discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate, and cost-effective genotyping method. With further test coverage to additionally include detection of other clinically significant HBA1/2 copy number variations, such as the Thai, Mediterranean, and Filipino deletions, LMS may eventually serve as a comprehensive method for large-scale thalassemia carrier screening.

PurposeEmerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted.MethodsA total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics.ResultsLow-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input.ConclusionIn the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.

Recurrent miscarriage (RM) affects millions of couples globally, and half of them have no demonstrated etiology. Genome sequencing (GS) is an enhanced and novel cytogenetic tool to define the contribution of chromosomal abnormalities in human diseases. In this study we evaluated its utility in RM-affected couples. We performed low-pass GS retrospectively for 1,090 RM-affected couples, all of whom had routine chromosome analysis. A customized sequencing and interpretation pipeline was developed to identify chromosomal rearrangements and deletions/duplications with confirmation by fluorescence in situ hybridization, chromosomal microarray analysis, and PCR studies. Low-pass GS yielded results in 1,077 of 1,090 couples (98.8%) and detected 127 chromosomal abnormalities in 11.7% (126/1,077) of couples; both members of one couple were identified with inversions. Of the 126 couples, 39.7% (50/126) had received former diagnostic results by karyotyping characteristic of normal human male or female karyotypes. Low-pass GS revealed additional chromosomal abnormalities in 50 (4.0%) couples, including eight with balanced translocations and 42 inversions. Follow-up studies of these couples showed a higher miscarriage/fetal-anomaly rate of 5/10 (50%) compared to 21/93 (22.6%) in couples with normal GS, resulting in a relative risk of 2.2 (95% confidence interval, 1.1 to 4.6). In these couples, this protocol significantly increased the diagnostic yield of chromosomal abnormalities per couple (11.7%) in comparison to chromosome analysis (8.0%, chi-square test p = 0.000751). In summary, low-pass GS identified underlying chromosomal aberrations in 1 in 9 RM-affected couples, enabling identification of a subgroup of couples with increased risk of subsequent miscarriage who would benefit from a personalized intervention.

PURPOSE Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared to population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Accumulating experimental evidence has shown that the aberrant expression of microRNAs (miRNAs) is involved in the development and progression of human cervical cancer. Previously, we identified miR-182 as an oncomiRNA in cervical cancer. However, the mechanism by which miR-182 is regulated and the interaction between human papillomavirus (HPV) and miR-182 in cervical cancer development remains unknown. In the present study, we explored the link between HPV E7 and miR-182 and verified that high-risk HPV E7 upregulated miR-182 expression through TGF-β/Smad4 signaling pathway in cervical cancer. By contrast, low-risk HPV E7 did not affect the expression of TGF-β and miR-182. Mechanistically, as high-risk HPV E7 bound to pRb, E2F was released from the complex and bound to the TGF-β promoter region, resulting in TGF-β overexpression. Furthermore, the Smad4 signaling pathway was activated upon TGF-β overexpression, which led to an interaction between Smad4 and the miR-182 promoter region, subsequently inducing the upregulation of miR-182 in both cervical cancer cells and the surrounding normal cells. In conclusion, this newly identified high-risk HPV E7/TGF-β/miR-182 regulatory network might inform the development of specific therapeutic strategies for cervical cancer.

The culture failure rate of conventional karyotyping in products of conception evacuated from early pregnancy loss by traditional electrical vacuum aspiration and/or dilationcurettage remains high. We aim to determine whether obtaining products of conception from early pregnancy loss via another evacuation approach, ultrasound-guided manual vacuum aspiration, could decrease the culture failure rate of karyotyping.For patients with early pregnancy loss, ultrasound-guided manual vacuum aspiration (Case group) and traditional electrical vacuum aspiration and/or dilationcurettage (Control group) were applied as surgical methods for pregnancy loss management respectively. The evacuated products of conception were subjected to cytogenetic karyotyping analysis. The primary outcome was the culture failure rate of karyotyping. Secondary outcomes included the chromosomal abnormality spectrum, maternal cell contamination, and complications from the manual vacuum aspiration procedure.For the case group, 132 products of conception were genetically analyzed by conventional karyotyping. The culture failure rate was significantly lower than that of the control cohort (2.3% vs 7.4%, p = 0.027). 65.2% of cases were abnormal within the detection scope of karyotyping. The euploid female to male ratio was 0.82 (18:22), suggesting a lower likelihood of maternal cell contamination. The efficacy in achieving complete evacuation was 99.6%. There were no significant complications from the procedure.Ultrasound-guided manual vacuum aspiration approach significantly decreased the culture failure rate of karyotyping and decreased maternal cell contamination tendency, enabling a high and accurate cytogenetic diagnosis. It is especially crucial when the cytogenetic analysis is required in the early pregnancy loss clinics.

Chromosomal microarray analysis is the gold standard for copy-number variant (CNV) detection in prenatal and postnatal diagnosis. We aimed to determine whether next-generation sequencing (NGS) technology could be an alternative method for CNV detection in routine clinical application. Genome-wide CNV analysis (>50 kb) was performed on a multicenter group of 570 patients using a low-coverage whole-genome sequencing pipeline. These samples were referred for chromosomal analysis; CNVs (i.e., pathogenic CNVs, pCNVs) were classified according to the American College of Medical Genetics and Genomics guidelines. Overall, a total of 198 abortuses, 37 stillbirths, 149 prenatal, and 186 postnatal samples were tested. Our approach yielded results in 549 samples (96.3%). In addition to 119 subjects with aneuploidies, 103 pCNVs (74 losses and 29 gains) were identified in 82 samples, giving diagnostic yields of 53.2% (95% confidence interval: 45.8, 60.5), 14.7% (5.0, 31.1), 28.5% (21.1, 36.6), and 30.1% (23.6, 37.3) in each group, respectively. Mosaicism was observed at a level as low as 25%. Patients with chromosomal diseases or microdeletion/microduplication syndromes were diagnosed using a high-resolution genome-wide method. Our study revealed the potential of NGS to facilitate genetic diagnoses that were not evident in the prenatal and postnatal groups. Genet Med 18 9, 940–948.

Summary Objectives To characterize at high resolution DNA methylation changes of cytokines which occur in the genome of macrophages in association with Mycobacterium tuberculosis (MTB) infection Methods We studied the methylation profiles of THP-1 derived macrophage cells infected with clinical MTB strains [Beijing/W & non-Beijing/W lineage, sensitive (INH S , RIF S ) & resistant (INH R , RIF R ) strains] and of host macrophages from MTB infected cohorts (active & latent patients) with the human methylation CpG islands microarrays. Results Methylated modification on the promoter sequences of cytokines and their receptors were found to be associated with MTB infection in a strain- and host-dependent manner. Our epigenetic analyses revealed that infection with Beijing/W MTB strains enhanced IL6R, IL4R and IL17R hyper-methylations in infected macrophages. Validation of IL6R methylated sequence confirmed that MTB infection induced DNA methylation of CpG67 region in the IL6R promoter. In addition, studies on the human macrophage methylation profiles from the patient cohorts indicated that the methylation rate of IL17 family members and related genes were significantly altered in patients with active MTB infections. Conclusions Our study offered novel insights into the epigenetic changes in the interaction of host macrophages in MTB infections and warrant further explorations into these changes in modulating the immune response in active and latent MTB infections.

Background Sizing of FMR1 trinucleotide repeats in the clinical laboratory requires the use of capillary sequencer by PCR, or by a labor intensive measurement using Southern blot method. Our aim was to validate an accurate and robust PCR assay for quantification of CGG repeats. Methods We performed an analytical and clinical validation of a new PCR-based method that utilizes a low-cost capillary electrophoresis instrument and the FragilEase™ reagent kit. First, analytical performance was demonstrated on 12 Coriell reference samples comprising normal through full mutations. Subsequently, a cohort of 112 archived clinical DNA samples, enriched for premutation and full mutations, was analyzed. Results All samples were amplified successfully. Quantification of repeat numbers was interpreted by the use of standards with known repeats. Twenty-five full-mutation samples were successfully amplified with the largest allele size measured at 1380 repeats. The repeat numbers from the new assay were concordant with those obtained with the reference method. The intra-assay (CV Conclusion This new PCR-based method is reproducible and capable of identifying all Fragile X alleles. It is an accurate and robust method that facilitates Fragile X testing in a broader spectrum of clinical laboratories.

Objectives The purposes of this study were to identify aberrantly expressed miRNAs and investigate their pathogenic roles in cervical cancer. Methods miRNA expression was assessed in cervical cancer cell lines, micro-dissected normal cervical epithelium cells and primary cervical carcinoma by TaqMan RT-PCR. Spatial expression of miR-182 in cervical carcinoma and normal cervix was explored by in situ hybridization. HeLa xenograft mice model was used for evaluation of the effect on tumor growth of miR-182 inhibitor. Western blot, flow cytometry and gene expression analysis were used for identification of the functional role of miR-182 in HeLa cells. Results Two up-regulated ( miR-182 and − 183 ) and nine down-regulated ( miR-211, 145, 223, 150, 142-5p, 328, 195, 199b, 142-3p ) microRNAs were consistently identified in cervical cancer cell lines. Further investigation confirmed the most up-regulated miRNA ( miR-182 ) was significantly elevated in primary cervical carcinoma and discovered a significant correlation between the increased expression of miR-182 and advanced stages of cervical cancer. In HeLa xenograft mouse model, we demonstrated that inhibition of the miR-182 could exert the effect of tumor growth regression. Western blot, flow cytometry and pathway analysis for the HeLa cells with miR-182 over/down-expression in vitro showed that miR-182 was involved in apoptosis and cell cycle pathways, it also associated with the regulation of FOXO1 . Conclusions Our findings indicated that miR-182 plays an onco-miRNA role in cervical cancer and its alteration is associated with cervical cancer pathogenesis by disrupting cell proliferation.

Brain malformations, particularly related to early brain development, are a clinically and genetically heterogeneous group of fetal neurological disorders. Fetal cerebral malformation, predominantly of impaired prosencephalic development namely agenesis of the corpus callosum and septo-optic dysplasia, is the main pathological feature in fetus, and causes prominent neurodevelopmental retardation, and associated with congenital facial anomalies and visual disorders. Differential diagnosis of brain malformations can be extremely difficult even through magnetic resonance imaging. Advances in genomic and molecular genetics technologies have led to the identification of the sonic hedgehog pathways and genes critical to the normal brain development. Molecular cytogenetic and genetic studies have identified numeric and structural chromosomal abnormalities as well as mutations in genes important for the etiology of fetal neurological disorders. In this review, we update the molecular genetics findings of three common fetal neurological abnormalities, holoprosencephaly, lissencephaly and agenesis of the corpus callosum, in an attempt to assist in perinatal and prenatal diagnosis.

Background Prenatal sonographic diagnosis of Optiz G/BBB syndrome is difficult because the common clinical features, such as hypertelorism, hypospadias and abnormalities of midline structures, including laryngotracheoesophageal defects, are subtle. Method Chromosomal microarray (CMA) analysis using a target enriched Fetal DNA Chip design was performed on the DNA of a fetus with congenital cardiac abnormalities. Results Fetal DNA chip revealed a 48Kb single copy number loss within chromosome region Xp22.2 (arr[hg18]Xp22.2(10,627,354–10,675,946)x0 mat). This deletion included the 3′ UTR region of the MID1 gene predicted to cause the X-linked Opitz G/BBB syndrome. Conclusions This case supports the use of CMA in prenatal diagnosis of fetuses with congenital heart disease. CMA allows prenatal diagnosis of genomic aberrations at a much higher resolution compared with conventional karyotyping, and such findings enable proper genetic counseling and decision making in the pregnancy.

Background Previous studies have identified that mutations in a few genes, including T-BOX5, NKX2-5, EVC and GATA4, are associated with atrial septal defect (ASD). Methods A family of three generations with 4 members who were affected with ASD was investigated. To exclude the presence of any sub-microscopic chromosomal imbalance, high-resolution 1M array-based comparative genomic hybridization (aCGH) was performed. SNaPShot was used to certify the specificity of the finding mutation in the other family members. The coding region of GATA4 and NKX2-5 genes was screened by sequencing in another 30 cases including 10 cases of ventricular septal defect (VSD), 10 cases of atrial septal defect (ASD), 8 cases of VSD combined with ASD and 2 cases of atrioventricular septal defects (AVSD). Results No pathogenic copy number variant was detected by aCGH in the four affected family members with ASD. A novel non-synonymous variant, c.839C>T (T280M) in GATA4, was identified and segregated with all the ASD patients within this Chinese family. Such mutation was absent in other family members or present among sporadic CHD patients. In addition, we identified a non-synonymous variant in the NKX2-5 gene (P257A) associated with one congenital heart disease patient with VSD. Both mutations were not identified among healthy controls. Conclusion T280M mutation of GATA4 is suggested to be associated with ASD in this Chinese family.

Intraocular pressure (IOP) elevation is an important cause of glaucoma. Animal models of ocular hypertension have been widely used to mimic glaucoma to investigate the mechanisms underlying retinal ganglion cell (RGC) death and search for possible cure. The aim of the present study was to examine the role of JAK/STAT pathway in RGC viability in normal condition or after acute IOP elevation. Retinal explants obtained from intact or IOP-elevated eyes were firstly used to examine the effect of the JAK/STAT pathway inhibitors, AG490 and Jak Inhibitor I, on RGC viability in vitro. The role of this signal pathway was further investigated and confirmed in vivo. AG490 and Jak Inhibitor I were applied into the left eye on days 3, 9, and 15 post 2-h IOP elevation at 110mmHg. Fluorescence dye Fluorogold was used to retrogradely label surviving RGCs. Because macrophage recruitment was seen in the IOP-elevated eyes after inhibition of this pathway, clodronate liposomes were used to remove phagocytic cells in the eye and examine the role of JAK/STAT pathway in RGC survival independent of macrophages. Activities and location of JAK/STAT pathway in the retina were examined using Western blotting and immunohistochemistry. We found that inhibition of JAK/STAT pathway did not affect RGC survival in the retinal explants derived from intact eye but caused RGC death in the retinal explants that were derived from IOP-elevated eye. Importantly, the detrimental effect of JAK/STAT pathway inhibition on RGC survival was also observed in vivo following acute IOP elevation, but not in intact eye. In addition, both in vitro and in vivo experiments confirmed a detrimental action of phagocytic cells following acute IOP elevation and the pathway inhibition. Compatible with what were observed in vivo, Western blotting and immunohistochemistry showed that JAK/STAT activities were not present in intact retina, but acute IOP elevation activated JAK/STAT pathway in the retina, in the regions of inner nuclear layer and ganglion cell layer, including RGCs. The IOP elevation-induced JAK/STAT activities were effectively abolished by intravitreal application of AG490. This study thus shows that (1) acute IOP elevation activates JAK/STAT pathway in RGCs, and (2) JAK/STAT pathway mediates RGC survival following IOP elevation but not under normal condition.

Purpose To investigate the penetration of ofloxacin and moxifloxacin into the aqueous and vitreous after topical administration one hour before vitrectomy surgery. Design Prospective, randomized, double-blind case series study. Methods Twenty-seven patients undergoing vitrectomy were randomized to receive either topical ofloxacin 0.3% or moxifloxacin 0.5% every 10 minutes for one hour before surgery. Aqueous and vitreous samples were obtained and analyzed using high-performance liquidation chromatography. Results The moxifloxacin aqueous (1.576 ± 0.745 μg/ml) and vitreous (0.225 ± 0.013 μg/ml) levels were significantly higher than the ofloxacin aqueous (0.816 ± 0.504 μg/ml) (P = .0009) and vitreous (0.225 ± 0.013 μg/ml) [P = .0054] levels, respectively. The mean moxifloxacin aqueous and vitreous levels exceeded the minimum inhibitory concentration for 90% of isolates (MIC90) for a wide variety of bacteria implicated in endophthalmitis. In contrast, the aqueous level of ofloxacin exceeded the MIC90 of only a few organisms. Conclusions Moxifloxacin applied every 10 minutes during the hour before vitrectomy penetrated the eye significantly better than ofloxacin.

We carried out a pharmacokinetic study to determine the levels and profiles of catechins in pregnant rats and their fetuses after ingestion of green tea extract (GTE). We measured total catechin levels after enzyme digestions. Dams, at 15.5 days of gestation, were fed with GTE and catechins were measured in the maternal plasma, placenta, and fetus 0, 0.5, 1, 2, 3, 5, 8, 10, 12, 16, and 20 h after maternal GTE intake. The pharmacokinetic changes were analyzed by non compartmental models. We found that maternal plasma concentrations of catechins were about 10 times higher than in placenta and 50-100 times higher than in the fetus. AUC and Cmax levels of (-)-epicatechin (EC) were the highest in plasma while the levels of (-)-epigallocatechin gallate (EGCG) were the highest in the placenta and the fetus. The exposure level of catechin derivatives was higher than the gallate derivatives in maternal plasma after normalization but reversed in the placenta and fetus. The absorption of epi-isomers in plasma was found to be more favorable than their non epi-isomer counterparts. EGCG had the highest level of exposure (AUC) and the highest Cmax in the fetus, implying it may have potential for in utero antioxidant protection.

Objectives: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. Design and methods: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. Results: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including αA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). Conclusions: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

To determine the aqueous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in patients with active polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and pathologic myopia.Prospective, comparative control study.Aqueous humors were collected from 32 eyes of 32 patients for either active PCV or CNV. Among them, 11 eyes had active and symptomatic PCV, 12 eyes had active CNV secondary to AMD, and nine eyes had active CNV of pathologic myopia. Levels of VEGF and PEDF were determined by commercially available enzyme-linked immunosorbent assay kits. A group of 10 aqueous samples from 10 patients who underwent cataract surgery without other ocular or systemic diseases comprised the controls.VEGF concentrations in aqueous humor were markedly increased in patients with PCV, CNV of AMD, and CNV of myopia when compared with the controls (analysis of variance [ANOVA], P.001). VEGF levels in eyes with PCV were, however, significantly lower than those of exudative AMD (P = .045). The PEDF levels were also significantly different among the groups (ANOVA, P = .001), and we observed increased levels in PCV, CNV of AMD, and CNV of myopia.VEGF and PEDF factors were coexpressed and increased with positive correlation in aqueous humor of eyes with active PCV. The different levels of both factors in eyes of PCV and AMD might suggest distinct clinical entities or different angiogenesis courses between PCV and AMD.

We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2'-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of the retinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.

Tea polyphenols are well known for their beneficial health effects that involve their anti-carcinogenic, anti-mutagenic, anti-pathogenic and anti-oxidative properties. The main polyphenols of green tea are favan-3-ols (catechins) and their corresponding gallate compounds, which constitute about one-third of the dry weight of tea leaves. Their main ingredients are (+)-catechin (C), (−)-epicatechin (EC), (−)-gallocatechin (GC), (−)-epigallocatechin (EGC), (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), (−)-gallocatechin gallate (GCG) and (−)-epigallocatechin gallate (EGCG). Each has slightly different biological properties. We have developed a method to simultaneously analyze all these compounds in plasma and urine. The samples were first incubated with β- d -glucuronidase and sulfatase to release the catechin residues from their corresponding conjugates for subsequent extraction by selective solid phase column, Waters Oasis HLB extraction cartridges. The extracted molecules were resolved by reversed phase HPLC and monitored by coulometric chemical detection on a CoulArray detector. All eight catechin compounds were analyzed in a single chromatogram within 25 min. For plasma and urine analyses, good linearity (>0.9950) was validated in the range 10–2000 and 10–5000 ng/ml, respectively. The coefficients of variance (CV) were less than 5%. Absolute recovery was greater than 85% and detection limit was 5 ng/ml. The chromatogram exhibited minimal interference as a result of the highly selective solid phase extraction and CoulArray detection.

Low density lipoprotein receptor related protein (LRP) is a receptor for protease complexes, and may function in cell growth and repair, and in tumor invasiveness. LRP expression increases in glioblastomas compared to lower grade astrocytomas. Two potential mechanisms for this increased expression were investigated. The LRP C766T polymorphism is protective against Alzheimer's disease, perhaps through alteration of LRP expression. The frequency of the polymorphism was measured in astrocytoma patients and controls, but no significant difference was found. Differential PCR revealed LRP gene amplification in four of 25 high-grade gliomas and 0 of 23 other brain tumors. Co-amplification with epidermal growth factor receptor (EGFR) occurred in all four of the LRP-amplified tumors. Thus, LRP amplification may be partly responsible for increased LRP expression in astrocytomas, and may often occur in conjunction with EGFR amplification.

sion was performed to control for confounding variables. RESULTS: 309 women had CVS performed and also had pregnancy outcomes available in our database. Absence of villi buds was associated with preterm birth (p .001) and GDM (p .005). 92.6% of samples had composite scores of 2-6. High composite score (7-9) was associated with gestational hypertension (p 0.038) and 5 minute Apgar less than 7 (p 0.032). Both high (7-9) and low (0-1) composite scores were associated with preterm delivery (p 0.002). Low composite score was associated with GDM (p 0.003). Gestational age at procedure was associated with morphology (p 0.00). The association of high or low composite morphology score with these outcomes remained significant in the logistic regression analysis. CONCLUSION: Morphologic characteristics of chorionic villi already demonstrate abnormal features in the late first trimester in some pregnancies destined for poorer obstetric outcomes. Identifying the underlying molecular etiology responsible for underor overdeveloped chorionic villi may aid in a greater understanding of the molecular mechanisms leading to these adverse pregnancy outcomes.