Reproductive Carrier Screening Results With Maternal Health Implications During Pregnancy.

OBJECTIVE: To identify conditions on a reproductive carrier screening panel with the potential for carrier manifestations during pregnancy and review the implications for obstetric care. METHODS: This was a retrospective cross-sectional study of consecutive samples from female patients aged 18-55 years submitted to a commercial laboratory for a 274-gene carrier screening panel (January 2020 to September 2022). A literature review was performed to identify genes on the panel with potential for pregnancy complications in carriers. Carrier expression and published recommendations for clinical management were reviewed. RESULTS: We identified 12 genes with potential for carrier manifestations during pregnancy based on reports in the literature: nine with manifestations irrespective of the fetal genetic status ( ABCB11 , COL4A3 , COL4A4 , COL4A5 , DMD , F9 , F11 , GLA , and OTC ) and three ( CPT1A , CYP19A1 , and HADHA ) with manifestations only if the fetus is affected by the condition. Manifestations included cardiomyopathy, hemorrhage, gestational hypertensive disorders, cholestasis of pregnancy, acute fatty liver, hyperammonemic crisis, and maternal virilization. Published recommendations for carrier management were identified for 11 of the 12 genes. Of 91,637 tests performed during the study period, a pathogenic or likely pathogenic variant was identified in 2,139 (2.3%), giving a carrier frequency for any of the 12 genes of 1 in 43 (95% CI 1/41-45) 1,826 (2.0%) of the study population were identified as carriers for one of the nine genes with the potential for carrier manifestations irrespective of an affected or unaffected fetus. CONCLUSION: Approximately 1 in 40 female patients were identified as carriers for a condition with potential for maternal manifestations in pregnancy, including some serious or even life-threatening complications. Obstetric care professionals should be aware of the possibility of pregnancy complications among carriers and the available recommendations for management. FUNDING SOURCE: This study was funded by Natera, Inc.

Assessment of an automated approach for variant interpretation in screening for monogenic disorders: A single-center study.

BACKGROUND: Automation has been introduced into variant interpretation, but it is not known how automated variant interpretation performs on a stand-alone basis. The purpose of this study was to evaluate a fully automated computerized approach. METHOD: We reviewed all variants encountered in a set of carrier screening panels over a 1-year interval. Observed variants with high-confidence ClinVar interpretations were included in the analysis; those without high-confidence ClinVar entries were excluded. RESULTS: Discrepancy rates between automated interpretations and high-confidence ClinVar entries were analyzed. Of the variants interpreted as positive (likely pathogenic or pathogenic) based on ClinVar information, 22.6% were classified as negative (variants of uncertain significance, likely benign or benign) variants by the automated method. Of the ClinVar negative variants, 1.7% were classified as positive by the automated software. On a per-case basis, which accounts for variant frequency, 63.4% of cases with a ClinVar high-confidence positive variant were classified as negative by the automated method. CONCLUSION: While automation in genetic variant interpretation holds promise, there is still a need for manual review of the output. Additional validation of automated variant interpretation methods should be conducted.

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1-V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Development and comparison of a minimally-invasive model of autologous clot pulmonary embolism in Sprague-Dawley and Copenhagen rats.

BACKGROUND: Experimental models of pulmonary embolism (PE) that produce pulmonary hypertension (PH) employ many different methods of inducing acute pulmonary occlusion. Many of these models induce PE with intravenous injection of exogenous impervious objects that may not completely reproduce the physiological properties of autologous thromboembolism. Current literature lacks a simple, well-described rat model of autlogous PE. OBJECTIVE: Test if moderate-severity autologous PE in Sprague-Dawley (SD) and Copenhagen (Cop) rats can produce persistent PH. METHODS: blood was withdrawn from the jugular vein, treated with thrombin-Ca++ and re-injected following pretreatment with tranexamic acid. Hemodynamic values, clot weights and biochemical measurements were performed at 1 and 5 days. RESULTS: Infusion of clot significantly increased the right ventricular peak systolic pressure to 45-55 mm Hg, followed by normalization within 24 hours in SD rats, and within 5 days in COP rats. Clot lysis was 95% (24 hours) and 97% (5 days) in SD rats and was significantly lower in COP rats (70%, 24 hours; 87% 5 days). Plasma D-dimer was elevated in surgical sham animals and was further increased 8 hours after pulmonary embolism. Neither strain showed a significant increase in bronchoalveolar chemotactic activity, myeloperoxidase activity, leukocyte infiltration, or chemokine accumulation, indicating that there was no significant pulmonary inflammation. CONCLUSIONS: Both SD and COP rats exhibited near complete fibrinolysis of autologous clot PE within 5 days. Neither strain developed persistent PH. Experimental models of PE designed to induce sustained PH and a robust inflammatory response appear to require significant, persistent pulmonary vascular occlusion.

Molecular diversity of a North Carolina wastewater treatment plant as revealed by pyrosequencing.

We report the results of pyrosequencing of DNA collected from the activated sludge basin of a wastewater treatment plant in Charlotte, NC. Using the 454-FLX technology, we generated 378,601 sequences with an average read length of 250.4 bp. Running the 454 assembly algorithm over our sequences yielded very poor assembly, with only 0.3% of our sequences participating in assembly of significant contigs. Of the 117 contigs greater than 500 bp long that were assembled, the most common annotations were to transposases and hypothetical proteins. Comparing our sequences to known microbial genomes showed nonspecific recruitment, indicating that previously described taxa are only distantly related to the most abundant microbes in this treatment plant. A comparison of proteins generated by translating our sequence set to translations of other sequenced microbiomes shows a distinct metabolic profile for activated sludge with high counts for genes involved in metabolism of aromatic compounds and low counts for genes involved in photosynthesis. Taken together, these data document the substantial levels of microbial diversity within activated sludge and further establish the great utility of pyrosequencing for investigating diversity in complex ecosystems.

Transcriptional profile of right ventricular tissue during acute pulmonary embolism in rats.

Acute pulmonary embolism (PE) is the third leading cause of cardiovascular death in the United States. Moderate to severe PE can cause pulmonary arterial hypertension (PH) with resultant right ventricular (RV) heart damage. The mechanisms leading to RV failure after PE are not well defined, although it is becoming clear that PH-induced inflammatory responses are involved. We previously demonstrated profound neutrophil-mediated inflammation and RV dysfunction during PE that was associated with increased expression of several chemokine genes. However, a complete assessment of transcriptional changes in RVs during PE is still lacking. We have now used DNA microarrays to assess the alterations in gene expression in RV tissue during acute PE/PH in rats. Key results were confirmed with real-time RT-PCR. Nine CC-chemokine genes (CCL-2, -3, -4, -6, -7, -9, -17, -20, -27), five CXC-chemokine genes (CXCL-1, -2, -9, -10, -16), and the receptors CCR1 and CXCR4 were upregulated after 18 h of moderate PE, while one C-chemokine (XCL-1) and one CXC-chemokine (CXCL-12) were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated increased expression of many inflammatory genes. There was also a major shift in the expression of components of metabolic pathways, including downregulation of fatty acid transporters and oxidative enzymes, a change in glucose transporters, and upregulation of stretch-sensing and hypoxia-inducible transcription factors. This pattern suggests an extensive shift in cardiac physiology favoring the expression of the "fetal gene program."

Loading of peptide on to MHC molecule is not essential for clearance of Cryptosporidium parvum.

Transgenic and knockout mice usefully model the mechanisms that result in the clearance of Cryptosporidium parvum from the gut. CD4+ cells, cells expressing MHC class II, and CD154/CD40 interactions are essential. Unexpectedly, AND RAG-/- and DO11.10 RAG-/- mice with single specificities of T cells successfully clear Cryptosporidium infection. Clearance is accompanied by activation of CD4+ cells in the MLN. The ability of T cells bearing receptors for apparently irrelevant and non-cross reactive antigens to activate and to clear infection is surprising. The requirement for class II MHC expression for Cryptosporidium clearance raises the alternative possibilities that (a) class II MHC is required to present a peptide that is loaded as a consequence of infection or (b) that the cytokine environment engendered by a Cryptosporidium infection allows affinity for self MHC to activate naive T cells. In order to test the hypothesis that peptide loading is necessary, we used A betaE alpha-/-Ii-/- mice that express a hybrid IA-IE MHC molecule. They also carry a transgene that makes an E alpha peptide while disruption of their invariant chain blocks the loading of a foreign peptide on to their MHC class II molecules. After oral gavage, the course of infection was followed by ELISA. CD4+ cells in the MLN of these mice were activated to express CD69 and the infection was cleared. We conclude that the loading of a Cryptosporidium or other infection-dependent peptide onto the MHC class II molecules of APCs is not necessary for clearance of Cryptosporidium. Instead the TcR affinity for self-MHC must suffice for T cell activation in the cytokine environment resulting from infection.