Next Generation Sequencing in Newborn Screening in the United Kingdom National Health Service.

Next generation DNA sequencing (NGS) has the potential to improve the diagnostic and prognostic utility of newborn screening programmes. This study assesses the feasibility of automating NGS on dried blood spot (DBS) DNA in a United Kingdom National Health Service (UK NHS) laboratory. An NGS panel targeting the entire coding sequence of five genes relevant to disorders currently screened for in newborns in the UK was validated on DBS DNA. An automated process for DNA extraction, NGS and bioinformatics analysis was developed. The process was tested on DBS to determine feasibility, turnaround time and cost. The analytical sensitivity of the assay was 100% and analytical specificity was 99.96%, with a mean 99.5% concordance of variant calls between DBS and venous blood samples in regions with ≥30× coverage (96.8% across all regions; all variant calls were single nucleotide variants (SNVs), with indel performance not assessed). The pipeline enabled processing of up to 1000 samples a week with a turnaround time of four days from receipt of sample to reporting. This study concluded that it is feasible to automate targeted NGS on routine DBS samples in a UK NHS laboratory setting, but it may not currently be cost effective as a first line test.

Tribbles-1 and -2 are tumour suppressors, down-regulated in human acute myeloid leukaemia.

Constitutive MAPK signalling is observed in approximately 50% of acute myeloid leukaemia (AML) cases. JNK activation in particular is associated with treatment failure in AML. Tribbles proteins (trb-1, trb-2 and trb-3) are potent negative regulators of MAPK pathways influencing apoptosis, differentiation and cell-cycle progression. Here we aimed to examine tribbles gene expression in AML and to characterise their role in leukaemic cells. A microarray dataset was interrogated for tribbles expression levels in AML cases and healthy controls. Myeloid cell proliferation and apoptosis were assayed in response to trb-1/trb-2 gene knockdown and overexpression, as well as a physical and functional interaction between trb and C/EBPalpha. Trb-2 expression was reduced in AML compared to healthy controls (correlating with nucleophosmin (NPM1) mutations), while low trb-1 expression was associated with inactive C/EBPalpha. In vitro assays indicated that trb-1/trb-2 are growth restrictive and pro-apoptotic in Me-1 cells, each capable of inhibiting JNK activation. JNK inactivation was itself associated with reduced Bcl-2 Ser70 phosphorylation, a residue which, when phosphorylated, maintains the anti-apoptotic activity of Bcl-2. Consistent with this, tribbles-mediated dephosphorylation of Bcl-2 Ser70 was associated with subsequent apoptosis. Trb-1/trb-2 transcription appeared to be moderately C/EBPalpha-responsive, and physical interaction between C/EBPalpha and trb-1/trb-2 was observed, suggesting a potential for auto-regulation of trb-1 and trb-2 transcription. In conclusion, we propose that trb-1 and trb-2 tumour suppressor activity may be abrogated in a proportion of AML patients. This may lead to enhanced cell survival, and therefore contribute to pathogenesis of the disease. Trb-1/trb-2 may, therefore, represent useful therapeutic targets for the treatment of AML in patients with dys-regulated trb activity.

Regulation of the human protein S gene promoter by liver enriched transcription factors.

Protein S is expressed in a number of tissue types, one of the most physiologically relevant being the liver. However, transcriptional control of protein S gene expression is poorly understood. We have characterised a 638 bp area in the 5' flanking region of the human protein S gene, spanning all 10 previously reported transcription initiation sites, which demonstrates promoter activity in the human liver-derived cell line HepG2. More refined reporter gene analysis of this region enabled the identification of three transcription initiation sites whose absence is associated with significantly reduced promoter activity, together with a number of positively and negatively acting transcriptional regulatory elements. Consistent with these findings, DNaseI footprinting analysis identified eleven sites (I-XI) from within this 638 bp region that show evidence of binding nuclear proteins. We present evidence to show that the liver-specific factors hepatocyte nuclear factor 1 (HNF1) and HNF4 bind regions of the protein S promoter, which lie within the identified protein binding sites V and VIII, respectively, and that HNF4 activates the protein S promoter. Reporter gene analysis suggests that members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors are potent activators of protein S gene transcription in HepG2 cells.

Oestrogenic repression of human coagulation factor VII expression mediated through an oestrogen response element sequence motif in the promoter region.

Reporter gene analysis of two regions of the human factor VII (FVII) gene promoter (residues -658 to -1 and -348 to -1, where +1 is the start site of translation) in the mammalian liver-derived cell line HepG2 showed reduced transcriptional activity in the presence of oestrogenic factors. This effect was independent of promoter polymorphic haplotype. Similar analysis using a smaller region of the promoter spanning residues -187 to -1 failed to show any evidence of oestrogenic suppression. Electrophoretic mobility shift assays and supershift assays using recombinant oestrogen receptor alpha and anti-oestrogen receptor antibody localized the sequence motif to which oestrogen receptor was binding to residues -225 to -212 of the FVII promoter. The lack of oestrogenic suppression in a reporter gene construct spanning residues -658 to -1 modified to abolish oestrogen receptor binding at this site, confirmed the functional significance of this motif. Although superficially similar to the classical oestrogen response element (ORE), comprising two half sites separated by three spacer nucleotides, the FVII ORE represents an alternative type of ORE in which the two half sites are separated by just two spacer nucleotides. EMSAs indicated that increasing spacer nucleotide number from two to three in the FVII ORE, or decreasing it from three to two in a consensus ORE sequence motif, had a small effect on the binding affinity for oestrogen receptor. These data correlate with and provide a plausible mechanism for the inverse relationship between FVII and oestradiol levels observed during the menstrual cycle.