Effects of supplementation with nondigestible carbohydrates on fecal calprotectin and on epigenetic regulation of SFRP1 expression in the large-bowel mucosa of healthy individuals.

BACKGROUND: Hyperactive Wnt signaling is frequently observed in colorectal cancer. Higher intakes of dietary fiber [nondigestible carbohydrates (NDCs)] and the fermentation product butyrate are protective against colorectal cancer and may exert their preventative effects via modulation of the Wnt pathway. OBJECTIVES: We investigated the effects of supplementing healthy individuals with 2 NDCs [resistant starch (RS) and polydextrose] on fecal calprotectin concentrations and Wnt pathway-related gene expression. In addition, we determined whether effects on secreted frizzled-related protein 1 (SFRP1) expression are mediated via the epigenetic mechanisms DNA methylation and microRNA expression. DESIGN: In a randomized, double-blind, placebo-controlled trial (the Dietary Intervention, Stem cells and Colorectal Cancer (DISC) Study), 75 healthy participants were supplemented with RS and/or polydextrose or placebo for 50 d in a 2 × 2 factorial design. Pre- and postintervention stool samples and rectal mucosal biopsies were collected and used to quantify calprotectin and expression of 12 Wnt-related genes, respectively. The expression of 10 microRNAs predicted to target SFRP1 was also quantified by quantitative reverse transcriptase-polymerase chain reaction, and DNA methylation was quantified at 7 CpG sites within the SFRP1 promoter region by pyrosequencing. RESULTS: NDC supplementation did not affect fecal calprotectin concentration. SFRP1 mRNA expression was reduced by both RS (P = 0.005) and polydextrose (P = 0.053). RS and polydextrose did not affect SFRP1 methylation or alter the expression of 10 microRNAs predicted to target SFRP1. There were no significant interactions between RS and polydextrose. CONCLUSIONS: RS and polydextrose supplementation did not affect fecal calprotectin concentrations. Downregulation of SFRP1 with RS and polydextrose could result in increased Wnt pathway activity. However, effects on Wnt pathway activity and downstream functional effects in the healthy large-bowel mucosa remain to be investigated. The DISC Study was registered at clinicaltrials.gov as NCT01214681.

Combined Count- and Size-Based Analysis of Maternal Plasma DNA for Noninvasive Prenatal Detection of Fetal Subchromosomal Aberrations Facilitates Elucidation of the Fetal and/or Maternal Origin of the Aberrations.

BACKGROUND: Noninvasive prenatal detection of fetal subchromosomal copy number aberrations (CNAs) can be achieved through massively parallel sequencing of maternal plasma DNA. However, when a mother herself is a carrier of a CNA, one cannot discern if her fetus has inherited the CNA. In addition, false-positive results would become more prevalent when more subchromosomal regions are analyzed. METHODS: We used a strategy that combined count- and size-based analyses of maternal plasma DNA for the detection of fetal subchromosomal CNAs in 7 target regions for 10 test cases. RESULTS: For the 5 cases in which CNAs were present only in the fetus, the size-based approach confirmed the aberrations detected by the count-based approach. For the 5 cases in which the mother herself carried an aberration, we successfully deduced that 3 of the fetuses had inherited the aberrations and that the other 2 fetuses had not inherited the aberrations. No false positives were observed in this cohort. CONCLUSIONS: Combined count- and size-based analysis of maternal plasma DNA permits the noninvasive elucidation of whether a fetus has inherited a CNA from its mother who herself is a carrier of the CNA. This strategy has the potential to improve the diagnostic specificity of noninvasive prenatal testing.

Whole-genome array CGH evaluation for replacing prenatal karyotyping in Hong Kong.

OBJECTIVE: To evaluate the effectiveness of whole-genome array comparative genomic hybridization (aCGH) in prenatal diagnosis in Hong Kong. METHODS: Array CGH was performed on 220 samples recruited prospectively as the first-tier test study. In addition 150 prenatal samples with abnormal fetal ultrasound findings found to have normal karyotypes were analyzed as a 'further-test' study using NimbleGen CGX-135K oligonucleotide arrays. RESULTS: Array CGH findings were concordant with conventional cytogenetic results with the exception of one case of triploidy. It was found in the first-tier test study that aCGH detected 20% (44/220) clinically significant copy number variants (CNV), of which 21 were common aneuploidies and 23 had other chromosomal imbalances. There were 3.2% (7/220) samples with CNVs detected by aCGH but not by conventional cytogenetics. In the 'further-test' study, the additional diagnostic yield of detecting chromosome imbalance was 6% (9/150). The overall detection for CNVs of unclear clinical significance was 2.7% (10/370) with 0.9% found to be de novo. Eleven loci of common CNVs were found in the local population. CONCLUSION: Whole-genome aCGH offered a higher resolution diagnostic capacity than conventional karyotyping for prenatal diagnosis either as a first-tier test or as a 'further-test' for pregnancies with fetal ultrasound anomalies. We propose replacing conventional cytogenetics with aCGH for all pregnancies undergoing invasive diagnostic procedures after excluding common aneuploidies and triploidies by quantitative fluorescent PCR. Conventional cytogenetics can be reserved for visualization of clinically significant CNVs.

Noninvasive prenatal molecular karyotyping from maternal plasma.

Fetal DNA is present in the plasma of pregnant women. Massively parallel sequencing of maternal plasma DNA has been used to detect fetal trisomies 21, 18, 13 and selected sex chromosomal aneuploidies noninvasively. Case reports describing the detection of fetal microdeletions from maternal plasma using massively parallel sequencing have been reported. However, these previous reports were either polymorphism-dependent or used statistical analyses which were confined to one or a small number of selected parts of the genome. In this report, we reported a procedure for performing noninvasive prenatal karyotyping at 3 Mb resolution across the whole genome through the massively parallel sequencing of maternal plasma DNA. This method has been used to analyze the plasma obtained from 6 cases. In three cases, fetal microdeletions have been detected successfully from maternal plasma. In two cases, fetal microduplications have been detected successfully from maternal plasma. In the remaining case, the plasma DNA sequencing result was consistent with the pregnant mother being a carrier of a microduplication. Simulation analyses were performed for determining the number of plasma DNA molecules that would need to be sequenced and aligned for enhancing the diagnostic resolution of noninvasive prenatal karyotyping to 2 Mb and 1 Mb. In conclusion, noninvasive prenatal molecular karyotyping from maternal plasma by massively parallel sequencing is feasible and would enhance the diagnostic spectrum of noninvasive prenatal testing.

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone inhibits skin cancer M21 cell proliferation through aberrant expression of microtubules and the cell cycle.

Microtubules are a proven target for anticancer drug development because they are critical for mitotic spindle formation and the separation of chromosomes at mitosis. 2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (HL66) induced cell death with the large cells and multiple micronuclei in M21 skin cancer cells. We demonstrated that HL66-induced cell death is caspase-independent and accompanied by the failure of cell cycle progression. Therefore, HL66-induced cell death may be a mitotic catastrophe. HL66 inhibits the dephosphorylation on Thr14 or Tyr15 of cyclin-dependent kinase (Cdk) 1 and the formation of Cdk1/cyclin B1 complex, which might be associated with cell cycle arrest at the S and G(2)/M phases. HL66 is an antimicrotubule agent by molecular modeling on the basis of ligand binding to tubulin molecule. Furthermore, we also demonstrated that HL66, like vinblastine, is a tubulin-destabilizing agent via microtubule disruption in M21 cells. These results describe a novel pharmacological property of HL66 as a microtubule inhibitor, which may make it an attractive new agent for the treatment of skin cancer.

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.

OBJECTIVES: To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. DESIGN: Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. SETTING: Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. PARTICIPANTS: 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. MAIN OUTCOME MEASURES: Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. RESULTS: Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. CONCLUSION: Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.