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BackgroundCirculating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.MethodsWhole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.ResultsTwo parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.ConclusionsThese data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

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Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests. Since the introduction of NIPT to detect trisomy 21 in late 2011, the technology has rapidly advanced to analyze other autosomal and sex chromosome aneuploidies, and now includes the detection of subchromosomal deletion and duplication events. Here we provide a brief overview of how noninvasive prenatal testing using next-generation sequencing is performed.

ZAP70 expression has been shown to be involved in enhanced signalling and more aggressive disease in a subset of CLL. Mechanisms regulating ZAP70 expression are unknown. We have shown previously that despite the absence of a 5’ CpG island, the methylation status of a small region of CpG dinucleotides (CpGs) correlates with the transcriptional state of the gene in both normal lymphocytes and B cell leukemias. Quantitative methylation analysis of 605 CpGs across the 28kb genomic region spanning ZAP70 was performed by MassARRAY on a panel of 17 CLL tumor cell samples, 4 lymphoid cell lines and B cell, T cell and myeloid cell samples pooled from 3 normal individuals. All samples showed hypermethylation of the gene body and of the gene’s two 3’ CpG islands. However, there was variability between samples in the methylation of 12 consecutive CpGs within a 1kb predicted promoter region (PPR), spanning the transcription start site (TSS) and in the methylation of 24 consecutive CpGs in an adjacent 1kb differentially methylated region (DMR), downstream of the TSS. The methylation of the PPR and DMR, together with the expression status of the samples, suggested four different states for the gene (Table 1). Table 1 - ZAP70 gene states defined by ZAP70 expression status and methylation of the PPR and DMR. MEAN CpG METHYLATION (%) SAMPLE ZAP70 EXPRESSION STATUS PPR DMR GENE STATE NAMALWA − 65 82 I B CELLS − 48 82 I MYELOID − 53 80 I CLL6 − 4 86 II CLL7 − 5 75 II CLL8 − 12 78 II CLL10 − 12 62 II CLL11 − 4 62 II CLL12 − 21 77 II HBL2 − 18 60 II CLL13 + 4 40 III CLL14 + 5 46 III CLL15 + 7 45 III CLL16 + 9 43 III CLL17 + 5 56 III NALM6 + 8 52 III CLL1 + 3 4 IV CLL2 + 3 4 IV CLL9 + 4 6 IV CLL4 + 3 8 IV CLL5 + 4 16 IV CLL3 + 4 17 IV JURKAT + 3 4 IV T CELLS + 9 13 IV Bisulphite cloning and sequencing of a PCR amplicon spanning an exon1 C/A SNP (rs2276645) and the PPR/DMR junction was performed together with cDNA pyrosequencing of rs2276645 on the five CLL tumor samples identified with gene state III. All samples showed allele specific methylation (ASM) of the A allele within the DMR and almost complete restriction of ZAP70 expression to the hypomethylated C allele. Bisulphite pyrosequencing of two DMR CpGs in purified leukocyte populations from these cases showed that ASM appears restricted to CLL cells, with hypermethylation and hypomethylation of the myeloid and T cells respectively (Table2). This suggests that while methylation of the DMR is sufficient for allele restriction, ASM does not result from imprinting and may be restricted to CLL tumor cells. Table 2 – Mean methylation of 2 DMR CpGs in leukocyte populations from CLL patients with known ASM of the DMR in their tumor cells. MYELOID CELLS CLL CELLS T CELLS PATIENT CD15 (%) METHYLATION(%) CD19 (%) METHYLATION (%) CD2 (%) METHYLATION (%) CLL13 83 90 98 59 71 21 CLL14 98 99 98 50 88 10 CLL15 88 84 93 45 85 24 CLL16 99 96 99 52 82 18 CLL17 92 89 98 49 90 22 Native chromatin immunoprecipitation (N-ChIP) using anti-AcH3, H3K14Ac and H3K14Me2 antibodies was performed on the 4 cell lines and tumor cells from CLLs 1, 2, 6, 7, 13 and 14 from the MassARRAY series. PCR for amplicons across the PPR and DMR showed the presence of all 3 histone modifications in ZAP70 expressing JURKAT and NALM6 cells but these modifications were absent in the ZAP70 negative NAMALWA and HBL2 cells. In contrast, all 6 CLL samples showed enrichment for all 3 modifications, regardless of gene state, suggesting an open, active/permissive chromatin structure, despite clear differences in methylation of the DMR. Further bisulphite pyrosequencing and N-ChIP of NAMALWA and HBL2 cells cultured for 6 days in the presence of 0.5μM Decitibine showed concomitant DMR demethyaltion, increased AcH3 within the DMR and up regulation of ZAP70 expression, all of which were reversed when the drug stimulus was removed. Taken together this data suggests that ZAP70 is regulated by epigenetic mechanisms, with the methylation status of a small DMR playing a key role, sufficient to differentiate the transcriptional activity of two alleles within a single cell. It is apparent that the gene is primed for expression in all CLL cells and that methylation of the DMR is part of the key switching process between active transcription and silencing. The differences in DMR methylation between an individual’s expressing T cells and CLL cells, suggests that differences may exist in the mechanism of regulation between T and B cells and raises the possibility that such differences could be exploited as targets for therapy.

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