Rapid RHD Zygosity Determination Using Digital PCR.

BACKGROUND: Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. METHODS: DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)2 to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR (LR-PCR), next-generation sequencing, and conventional Sanger sequencing. RESULTS: The results showed clear and reliable determination of RHD zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as R1R2 (DCe/DcE), were R2r' (DcE/dCe) for 729M and R1r″ (DCe/dcE), R0ry (Dce/dCE), or RZr (DCE/dce) for 351D, in concordance with the digital PCR data. CONCLUSIONS: Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal RH zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous RHD-positive individuals.

Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR.

BACKGROUND: Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping. METHODS: Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS: The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS: qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.

Next-generation sequencing of 35 RHD variants in 16 253 serologically D- pregnant women in the Finnish population.

Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD- women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD- Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.

Correction to: Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

An amendment to this paper has been published and can be accessed via the original article.

Characterising a human endogenous retrovirus(HERV)-derived tumour-associated antigen: enriched RNA-Seq analysis of HERV-K(HML-2) in mantle cell lymphoma cell lines.

BACKGROUND: The cell-surface attachment protein (Env) of the HERV-K(HML-2) lineage of endogenous retroviruses is a potentially attractive tumour-associated antigen for anti-cancer immunotherapy. The human genome contains around 100 integrated copies (called proviruses or loci) of the HERV-K(HML-2) virus and we argue that it is important for therapy development to know which and how many of these contribute to protein expression, and how this varies across tissues. We measured relative provirus expression in HERV-K(HML-2), using enriched RNA-Seq analysis with both short- and long-read sequencing, in three Mantle Cell Lymphoma cell lines (JVM2, Granta519 and REC1). We also confirmed expression of the Env protein in two of our cell lines using Western blotting, and analysed provirus expression data from all other relevant published studies. RESULTS: Firstly, in both our and other reanalysed studies, approximately 10% of the transcripts mapping to HERV-K(HML-2) came from Env-encoding proviruses. Secondly, in one cell line the majority of the protein expression appears to come from one provirus (12q14.1). Thirdly, we find a strong tissue-specific pattern of provirus expression. CONCLUSIONS: A possible dependency of Env expression on a single provirus, combined with the earlier observation that this provirus is not present in all individuals and a general pattern of tissue-specific expression among proviruses, has serious implications for future HERV-K(HML-2)-targeted immunotherapy. Further research into HERV-K(HML-2) as a possible tumour-associated antigen in blood cancers requires a more targeted, proteome-based, screening protocol that will consider these polymorphisms within HERV-K(HML-2). We include a plan (and necessary alignments) for such work.

Dietary intake of inorganic nitrate in vegetarians and omnivores and its impact on blood pressure, resting metabolic rate and the oral microbiome.

Vegetarian diets are commonly associated with lower blood pressure levels. This has been related to greater consumption of inorganic nitrate, since vegetables are the main source of this anion. Dietary nitrate is reduced to nitrite by commensal bacteria in the mouth, which in turn leads to increased circulatory nitrite availability. Nitrite can form nitric oxide by several pathways promoting a reduction in the vascular tone and lower blood pressure. This study tested whether vegetarians have higher concentrations of nitrite in saliva and plasma, and lower blood pressure and resting metabolic rate (RMR), due to higher intakes of nitrate, compared to omnivores. Following a non-randomized, cross-over and single-blinded design we measured dietary nitrate intake, blood pressure and RMR in young and healthy vegetarians (n = 22) and omnivores (n = 19) with similar characteristics after using placebo or antibacterial mouthwash for a week to inhibit oral bacteria. Additionally, we analyzed salivary and plasma nitrate and nitrite concentrations, as well as the oral nitrate-reduction rate and oral microbiome in both groups. Dietary nitrate intake in vegetarians (97 ±â€¯79 mg/day) was not statistically different (P > 0.05) to omnivores (78 ±â€¯47 mg/day). Salivary and plasma nitrate and nitrite concentrations were similar after placebo mouthwash in both groups (P > 0.05). The oral nitrate-reducing capacity, abundance of oral bacterial species, blood pressure and RMR were also similar between vegetarians and omnivores (P > 0.05). Antibacterial mouthwash significantly decreased abundance of oral nitrate-reducing bacterial species in vegetarians (_16.9%; P < 0.001) and omnivores (_17.4%; P < 0.001), which in turn led to a significant reduction of the oral nitrate-reducing capacity in vegetarians (-78%; P < 0.001) and omnivores (-85%; P < 0.001). However, this did not lead to a significant increase in blood pressure and RMR in either groups (P > 0.05). These findings suggest that vegetarian diets may not alter nitrate and nitrite homeostasis, or the oral microbiome, compared to an omnivore diet. Additionally, inhibition of oral nitrite synthesis for a week with antibacterial mouthwash did not cause a significant raise in blood pressure and RMR in healthy, young individuals independent of diet.