Performance of a cell-free DNA prenatal screening test, choice of prenatal procedure, and chromosome conditions identified during pregnancy after low-risk cell-free DNA screening.

OBJECTIVES: To evaluate the performance of cell-free DNA (cfDNA) screening for common fetal aneuploidies, choice of prenatal procedure, and chromosome conditions identified during pregnancy after low-risk cfDNA screening. METHOD: A single-center prenatal cfDNA screening test was employed to detect trisomies 21, 18, and 13 (T21, T18, T13) and sex chromosome aneuploidies (SCAs). Test performance, choice of prenatal procedure, and cytogenetic results in pregnancies with low-risk cfDNA screening were reviewed. RESULTS: CfDNA screening of 38,289 consecutive samples identified 720 (1.9%) pregnancies at increased risk for aneuploidy. Positive predictive values (PPVs) for high-risk singleton pregnancies were 98.5% (T21), 92.5% (T18) and 55.2% (T13). PPVs for SCAs ranged from 30.6% to 95.2%. Most women elected chorionic villus sampling for prenatal diagnosis of T21, T18 and T13; amniocentesis and/or postnatal testing were commonly chosen for SCAs. Cytogenetic tests from 616 screen-negative pregnancies identified 64 cases (12.7%) with chromosome conditions not detected by cfDNA screening, including triploidy (n = 30) and pathogenic and likely pathogenic copy number variants (n = 34). A further 15 (0.04%) false-negative common aneuploidy results were identified. CONCLUSIONS: CfDNA screening was highly accurate for detecting fetal aneuploidy in this general-risk obstetric population. Fetal ultrasound and prenatal diagnostic testing were important in identifying chromosome conditions in pregnancies screened as low-risk.

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke.

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.

Immunopathogenesis, loss of T cell tolerance and genetics of autoimmune gastritis.

Over the past 10 years experimental autoimmune gastritis has been established as a highly defined model of organ-specific autoimmunity. Autoimmune gastritis represents one of the few autoimmune diseases in which the causative autoantigens, namely the gastric H/K ATPase alpha- and beta-subunits, are defined. Furthermore, it has been clearly established that a CD4+ T cell response to the H/K ATPase beta-subunit, in particular, is essential for the initiation of autoimmune gastritis. The immunopathology of autoimmune gastritis is due to a disruption of the normal developmental pathways of the mucosa, rather than a direct depletion of the end-stage parietal and zymogenic cells. CD4+CD25+ regulatory T cells were first described in experimental autoimmune gastritis and there has been a recent explosion of interest in the potential role of these immunoregulatory T cells in protection against a variety of autoimmune diseases. The availability of H/K ATPase deficient mice has begun to provide considerable insight into the basis for tolerance to the gastric autoantigens. Experimental autoimmune gastritis has also provided valuable insight into our understanding of the genetics of disease susceptibility and four distinct genetic regions have been identified which confer susceptibility to this organ-specific disease. The highlights of these recent advances are the subject of this review.

A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock-in mice.

The human serpin, proteinase inhibitor 6 (PI-6/SERPINB6), is a protease inhibitor expressed in many tissues. It inhibits a large number of proteases, including cathepsin G in granulocytes and monocytes. To determine the temporal and spatial distribution of PI-6, mice were generated in which exon 2 of the PI-6 ortholog SPI3 (Serpinb6) was replaced with a green fluorescent protein (GFP) reporter gene. This placed GFP under the control of the regulatory elements and initiation codon of the SPI3 gene. The neomycin selection cassette was flanked by loxP sites to allow excision from the targeted allele. GFP expression in heterozygous and SPI3-deficient mice accurately reflected the tissue distribution of SPI3 in all organs tested and allowed precise comparisons of expression levels. Interestingly, retention of the neomycin cassette in targeted mice resulted in 2-10-fold increases of GFP in leukocytes, but without affecting tissue-specific expression patterns. This is the first example of selection cassette retention specifically increasing reporter gene expression in targeted mice and reinforces the view that selection cassettes must be removed to avoid confounding effects on reporter gene expression patterns.

Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse.

Ov-serpins are intracellular proteinase inhibitors implicated in the regulation of tumor progression, inflammation, and cell death. The 13 human ov-serpin genes are clustered at 6p25 (3 genes) and 18q21 (10 genes), and share common structures. We show here that a 1-Mb region on mouse chromosome 13 contains at least 15 ov-serpin genes compared with the three ov-serpin genes within 0.35 Mb at human 6p25 (SERPINB1 (MNEI), SERPINB6 (PI-6), SER-PINB9 (PI-9)). The mouse serpins have characteristics of functional inhibitors and fall into three groups on the basis of similarity to MNEI, PI-6, or PI-9. The genes map between the mouse orthologs of the Werner helicase interacting protein and NAD(P)H menadioine oxidoreductase 2 genes, in a region that contains the markers D13Mit136 and D13Mit116. They have the seven-exon structure typical of human 6p25 ov-serpin genes, with identical intron phasing. Most show restricted patterns of expression, with common sites of synthesis being the placenta and immune tissue. Compared with human, this larger mouse serpin repertoire probably reflects the need to regulate a larger proteinase repertoire arising from differing evolutionary pressures on the reproductive and immune systems.