Specifying a Gold Standard for the Validation of Fetal Fraction Estimation in Prenatal Screening.

BACKGROUND: An estimate of fetal fraction (FF) is needed for DNA-based screening for trisomy 21 and other aneuploidies, but there is no gold standard to validate FF measurement methods. We specify a gold standard and use it to validate a method of measuring FF (SeqFF) in singleton pregnancies. METHODS: The gold standard was a formula derived from 2 elements: (a) an estimate of the percentage of DNA fragments in maternal plasma from chromosome 21 (%Ch21) in pregnancies without trisomy 21, 18, or 13 (PU) and (b) calculation of %Ch21 with increasing FF in trisomy 21 pregnancies (P21). The SeqFF method was evaluated by plotting regression lines of %Ch21 and SeqFF estimates of FF in 31 singleton male and 31 female trisomy 21 pregnancies and comparing the regressions with the reference line derived from the gold standard formula. RESULTS: The gold standard formula was P21 = (1/2)PUFF + PU, with FF expressed as a proportion, or converting %Ch21 to multiples of the median (MoM), P21(MoM) = (1/2)FF + 1. Based on 3865 pregnancies, the PU was 1.2935%. The regression lines for trisomy 21 pregnancies with male and female fetuses were almost identical to the gold standard reference line (regression slopes in MoMs 0.52 and 0.50, respectively, compared with 0.50 for the gold standard reference line). CONCLUSIONS: The proposed gold standard can be used to validate different methods of estimating FF in singleton pregnancies. SeqFF is an accurate method of estimating FF.

The IONA® Test: Development of an Automated Cell-Free DNA-Based Screening Test for Fetal Trisomies 13, 18, and 21 That Employs the Ion Proton Semiconductor Sequencing Platform.

OBJECTIVE: To develop a screening test for fetal trisomy 13, 18, and 21 using cell-free DNA from maternal blood with an automated workflow using the Ion Proton sequencing platform. METHODS: An automated next-generation sequencing workflow was developed using the Ion Proton sequencing platform and software developed for straightforward bioinformatic analysis. An algorithm was developed using 239 samples to determine the likelihood of trisomy, using DNA fragment counts and a fetal fraction validity check; the results were compared with those from invasive diagnostic procedures. A further 111 samples were used to assess the tests' sensitivity (detection rate) and specificity (1 minus false-positive rate). RESULTS: The 110 of a possible 111 valid samples used to verify the IONA® test gave 100% sensitivity and specificity, compared with invasive diagnostic procedures; one failed the fetal fraction validity check giving a sample failure rate of 0.29% across all 350 analysed samples. CONCLUSION: The data indicate that the IONA test provides a robust, accurate automated workflow suitable for use on maternal blood samples to screen for trisomies 13, 18, and 21. The test has the potential to reduce the number of unnecessary invasive procedures performed and facilitate testing by screening laboratories.

A simple method to allow for guanine-cytosine amplification error in prenatal DNA screening for trisomy 18.

BACKGROUND: A source of error in prenatal screening for trisomies is PCR amplification error associated with guanine-cytosine (GC) content of DNA fragments in maternal plasma. We describe a simple method of allowing for this. METHODS: Data from a Reflex DNA screening programme (67 trisomy 18 and 83 unaffected pregnancies) were used to compare the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts (because chromosome 8 has a similar GC content to chromosome 18) with the percentage of chromosome 18 DNA counts using counts from all autosomes in the denominator, with and without an all autosome correction for the GC content of the DNA fragments. RESULTS: A chromosome 18 to 8 ratio of DNA fragment counts was more discriminatory than the percentage of all autosome counts arising from chromosome 18 without, or with an all autosome correction for GC content bias. It achieves a high screening performance, eg. for a 0.25% false-positive rate, a 97% detection rate instead of 49% without a correction for GC content, and 91% with an all autosome correction for GC content. CONCLUSION: Consideration can be given to using the ratio of chromosome 18 DNA fragment counts to chromosome 8 DNA fragment counts in cell-free DNA prenatal screening for trisomy 18, avoiding the need for more complex methods of making a correction for the GC content currently used.

Prenatal maternal plasma DNA screening for cystic fibrosis: A computer modelling study of screening performance.

Background: Prenatal cystic fibrosis (CF) screening is currently based on determining the carrier status of both parents. We propose a new method based only on the analysis of DNA in maternal plasma. Methods: The method relies on the quantitative amplification of the CF gene to determine the percentage of DNA fragments in maternal plasma at targeted CF mutation sites that carry a CF mutation. Computer modelling was carried out to estimate the distributions of these percentages in pregnancies with and without a fetus affected with CF. This was done according to the number of DNA fragments counted and fetal fraction, using the 23 CF mutations recommended by the American College of Medical Genetics for parental carrier testing. Results: The estimated detection rate (sensitivity) is 70% (100% of those detected using the 23 mutations), the false-positive rate 0.002%, and the odds of being affected given a positive screening result 14:1, compared with 70%, 0.12%, and 1:3, respectively, with current prenatal screening based on parental carrier testing. Conclusions: Compared with current screening practice based on parental carrier testing, the proposed method would substantially reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without reducing the CF detection rate. The expected advantages of the proposed method justify carrying out the necessary test development for use in a clinical validation study.

Promoter analysis of human corticotropin-releasing factor (CRF) type 1 receptor and regulation by CRF and urocortin.

We report the full genomic organization of the human gene for the corticotropin-releasing factor (CRF) receptor type 1 (CRFR1), with complete mapping of exons 1-14. The 5' flanking region (2.4 kb) of the gene encoding for human CRFR1 was isolated, sequenced, and characterized. Two major transcriptional start sites were determined at -265 and -238, relative to the ATG start site (+1). Transient expression of constructs containing sequentially deleted 5'-flanking sequences of CRFR1 fused to luciferase, revealed the minimal promoter sequence 370 bp in size, as shown by assays in neuroblastoma (SH-5YSY), teratocarcinoma (NT2), and adenocarcinoma (MCF 7) cell lines. CRF and UCN markedly increased promoter activity during transient CRFR1 expression studies. Similarly, CRF and UCN up-regulate the endogenous CRFR1 at the mRNA level in NT2 and MCF 7 cells. To dissect further the mechanisms involved, we have used primary myometrial cells transfected with the CRFR1 promoter. CRF and UCN increased the promoter activity, an effect blocked by protein kinase (PK)A and PKC inhibitors. Both CRF and UCN cause a positive feedback effect in primary cultures of human pregnant myometrial cells, by increasing mRNA expression of CRFR1. This effect appears to be dependent on activation of both PKA and PKC by CRF, whereas UCN's effect was mediated solely via PKC activation. Collectively, our data suggest that the CRFR1 gene is under the influence of both CRF and UCN, acting via distinct signaling pathways to create a positive feedback loop and regulate further the transcription of the receptor.

Non-ß-cell progenitors of ß-cells in pregnant mice.

Pregnancy is a normal physiological condition in which the maternal ß-cell mass increases rapidly about two-fold to adapt to new metabolic challenges. We have used a lineage tracing of ß-cells to analyse the origin of new ß-cells during this rapid expansion in pregnancy. Double transgenic mice bearing a tamoxifen-dependent Cre-recombinase construct under the control of a rat insulin promoter, together with a reporter Z/AP gene, were generated. Then, in response to a pulse of tamoxifen before pregnancy, ß-cells in these animals were marked irreversibly and heritably with the human placental alkaline phosphatase (HP AP). First, we conclude that the lineage tracing system was highly specific for ß-cells. Secondly, we scored the proportion of the ß-cells marked with HP AP during a subsequent chase period in pregnant and non-pregnant females. We observed a dilution in this labeling index in pregnant animal pancreata, compared to nonpregnant controls, during a single pregnancy in the chase period. To extend these observations we also analysed the labeling index in pancreata of animals during the second of two pregnancies in the chase period. The combined data revealed statistically-significant dilution during pregnancy, indicating a contribution to new beta cells from a non-ß-cell source. Thus for the first time in a normal physiological condition, we have demonstrated not only ß-cell duplication, but also the activation of a non-ß-cell progenitor population. Further, there was no transdifferentiation of ß-cells to other cell types in a two and half month period following labeling, including the period of pregnancy.

Unequal representation of different unique genomic DNA sequences in the cell-free plasma DNA of individual donors.

OBJECTIVES: To assess whether different genomic cell-free DNAs are equally abundant in the plasma of individual donors, and any relationship between DNA methylation and representation in plasma. DESIGN AND METHODS: The concentrations of DNA in plasma were determined by real-time PCR. RESULTS: Different DNA sequences were not equally represented. The relative abundances were similar in different donors. CONCLUSIONS: Different DNA sequences are not equally abundant in plasma, with no relationship between DNA methylation and abundance.

Methylation of the imprinted GNAS1 gene in cell-free plasma DNA: equal steady-state quantities of methylated and unmethylated DNA in plasma.

BACKGROUND: Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. METHODS: We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. RESULTS: Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. CONCLUSIONS: Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults.

Noninvasive prenatal diagnosis of aneuploidy using cell-free nucleic acids in maternal blood: promises and unanswered questions.

The discovery of cell-free fetal (cff) DNA and RNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis (NIPD) for the past decade. Detection of paternally derived alleles in cff DNA is becoming well established. Now much interest is focussing on NIPD of fetal chromosomal abnormalities, such as trisomy 21, which is a considerable challenge because this demands accurate quantitative measurements of the amounts of specific cff DNA or cff RNA sequences in maternal blood samples. Emerging strategies for distinguishing and quantifying the fetal nucleic acids in the maternal circulation promise continued development of the field, and pose a number of unanswered questions.

Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome.

This report describes the first identification and characterization of three chromosome-21-specific DNA sequences (and reference sequences from other chromosomes) that are differentially methylated between peripheral blood and placental tissue, with the aim of providing epigenetic biomarkers for quantifying cell-free fetal DNA in maternal plasma. To select sequences to be screened for differential methylation, three strategies were adopted: (i) investigating promoters of highly differentially expressed genes; (ii) choosing 'random' promoter regions; and (iii) choosing 'random' non-promoter regions. Over 200 pre-selected DNA sequences were screened using a methylation-specific restriction enzyme assay. Differentially methylated sequences located at 21q22.3 (AIRE, SIM2 and ERG genes), 1q32.1 (CD48 gene and FAIM3 gene), 2p14 (ARHGAP25 gene) and 12q24 (SELPLG gene) were identified. Bisulphite conversion confirmed that CpG sites within the AIRE promoter region are highly differentially methylated, and optimized methylation-specific primers for this region that are highly specific for placental DNA were devised. Next, it was shown that the methylation status of chorionic villus sample DNA from first trimester pregnancies matched the hypermethylated state of term placenta. Thus there is no indication of a difference in methylation status between early and term pregnancy for the sequences tested. The identified sequences constitute candidate biomarkers for non-invasive prenatal diagnosis of Down syndrome.

Analysis of Fcgamma receptor haplotypes in rheumatoid arthritis: FCGR3A remains a major susceptibility gene at this locus, with an additional contribution from FCGR3B.

The Fcgamma receptors play important roles in the initiation and regulation of many immunological and inflammatory processes, and genetic variants (FCGR) have been associated with numerous autoimmune and infectious diseases. The data in rheumatoid arthritis (RA) are conflicting and we previously demonstrated an association between FCGR3A and RA. In view of the close molecular proximity with FCGR2A, FCGR2B and FCGR3B, additional polymorphisms within these genes and FCGR haplotypes were examined to refine the extent of association with RA. Biallelic polymorphisms in FCGR2A, FCGR2B and FCGR3B were examined for association with RA in two well characterized UK Caucasian and North Indian/Pakistani cohorts, in which FCGR3A genotyping had previously been undertaken. Haplotype frequencies and linkage disequilibrium were estimated across the FCGR locus and a model-free analysis was performed to determine association with RA. This was followed by regression analysis, allowing for phase uncertainty, to identify the particular haplotype(s) that influences disease risk. Our results reveal that FCGR2A, FCGR2B and FCGR3B were not associated with RA. The haplotype with the strongest association with RA susceptibility was the FCGR3A-FCGR3B 158V-NA2 haplotype (odds ratio 3.18, 95% confidence interval 1.13-8.92 [P = 0.03] for homozygotes compared with all genotypes). The association was stronger in the presence of nodules (odds ratio 5.03, 95% confidence interval 1.44-17.56; P = 0.01). This haplotype was also more common in North Indian/Pakistani RA patients than in control individuals, but not significantly so. Logistic regression analyses suggested that FCGR3A remained the most significant gene at this locus. The increased association with an FCGR3A-FCGR3B haplotype suggests that other polymorphic variants within FCGR3A or FCGR3B, or in linkage disequilibrium with this haplotype, may additionally contribute to disease pathogenesis.