Clinical Perspective on Use of Long-Read Sequencing in Prenatal Diagnosis of Thalassemia.

This is an editorial focusing on the clinical perspective of a long-read sequencing method in the prenatal diagnosis of alpha- and beta-thalassemia, including a comparison between this method and standard PCR-based methods. Though incremental, the increased sensitivity and specificity using long-read sequencing is an important advantage of this methodology in the prenatal diagnostic arena due to false positive or false negative results having greater consequence when a family is making decisions about their pregnancy.

A genomic meta-analysis of clinical variables and their association with intrinsic molecular subsets in systemic sclerosis.

OBJECTIVES: Four intrinsic molecular subsets (inflammatory, fibroproliferative, limited, normal-like) have previously been identified in SSc and are characterized by unique gene expression signatures and pathways. The intrinsic subsets have been linked to improvement with specific therapies. Here, we investigated associations between baseline demographics and intrinsic molecular subsets in a meta-analysis of published datasets. METHODS: Publicly available gene expression data from skin biopsies of 311 SSc patients measured by DNA microarray were classified into the intrinsic molecular subsets. RNA-sequencing data from 84 participants from the ASSET trial were used as a validation cohort. Baseline clinical demographics and intrinsic molecular subsets were tested for statistically significant associations. RESULTS: Males were more likely to be classified in the fibroproliferative subset (P = 0.0046). SSc patients who identified as African American/Black were 2.5 times more likely to be classified as fibroproliferative compared with White/Caucasian patients (P = 0.0378). ASSET participants sera positive for anti-RNA pol I and RNA pol III autoantibodies were enriched in the inflammatory subset (P = 5.8 × 10-5, P = 9.3 × 10-5, respectively), while anti-Scl-70 was enriched in the fibroproliferative subset. Mean modified Rodnan Skin Score (mRSS) was statistically higher in the inflammatory and fibroproliferative subsets compared with normal-like (P = 0.0027). The average disease duration for inflammatory subset was less than fibroproliferative and normal-like intrinsic subsets (P = 8.8 × 10-4). CONCLUSIONS: We identified multiple statistically significant differences in baseline demographics between the intrinsic subsets that may represent underlying features of disease pathogenesis (e.g. chronological stages of fibrosis) and have implications for treatments that are more likely to work in certain SSc populations.

Macrophages in Systemic Sclerosis: Novel Insights and Therapeutic Implications.

PURPOSE OF REVIEW: Macrophages play key roles in tissue homeostasis and immune surveillance, mobilizing immune activation in response to microbial invasion and promoting wound healing to repair damaged tissue. However, failure to resolve macrophage activation can lead to chronic inflammation and fibrosis, and ultimately to pathology. Activated macrophages have been implicated in the pathogenesis of systemic sclerosis (SSc), although the triggers that induce immune activation in SSc and the signaling pathways that underlie aberrant macrophage activation remain unknown. RECENT FINDINGS: Macrophages are implicated in fibrotic activation in SSc. Targeted therapeutic interventions directed against SSc macrophages may ameliorate inflammation and fibrosis. While current studies have begun to elucidate the role of macrophages in disease initiation and progression, further work is needed to address macrophage subset heterogeneity within and among SSc end-target tissues to determine the disparate functions mediated by these subsets and to identify additional targets for therapeutic intervention.

Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased α-SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-ß1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

Analytical validation of a semi-automated methodology for quantitative measurement of SARS-CoV-2 RNA in wastewater collected in northern New England.

Wastewater-based epidemiology (WBE) can be used to monitor the community presence of infectious disease pathogens of public health concern such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral nucleic acid has been detected in the stool of SARS-CoV-2-infected individuals. Asymptomatic SARS-CoV-2 infections make community monitoring difficult without extensive and continuous population screening. In this study, we validated a procedure that includes manual pre-processing, automated SARS-CoV-2 RNA extraction and detection workflows using both reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR). Genomic RNA and calibration materials were used to create known concentrations of viral material to determine the linearity, accuracy, and precision of the wastewater extraction and SARS-CoV-2 RNA detection. Both RT-qPCR and RT-ddPCR perform similarly in all the validation experiments, with a limit of detection of 50 copies/mL. A wastewater sample from a care facility with a known outbreak was assessed for viral content in replicate, and we showed consistent results across both assays. Finally, in a 2-week survey of two New Hampshire cities, we assessed the suitability of our methods for daily surveillance. This paper describes the technical validation of a molecular assay that can be used for long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.IMPORTANCEThis paper describes the technical validation of a molecular assay that can be used for the long-term monitoring of SARS-CoV-2 in wastewater as a potential tool for community surveillance to assist with public health efforts.

Wastewater-Based SARS-CoV-2 Surveillance in Northern New England.

SARS-CoV-2 viral RNA is shed in the stool of 55-70% of infected individuals and can be detected in community wastewater up to 7 days before people present with COVID-19 symptoms. The detection of SARS-CoV-2 RNA in wastewater may serve as a lead indicator of increased community transmission. Here, we monitored viral concentrations in samples collected from nine municipal wastewater facilities in New Hampshire (NH) and Vermont (VT).Twenty-four-h composite primary influent wastewater samples were collected from nine municipal wastewater treatment facilities twice per week for 5 months (late September 2020 to early February 2021). Wastewater was centrifuged for 30 min at 4600 × g, then the supernatant was frozen until further analysis. Once thawed, samples were concentrated, extracted, and tested for SARS-CoV-2 RNA using reverse transcriptase-quantitative PCR (RT-qPCR) and reverse transcriptase-droplet digital PCR (RT-ddPCR) detection methods. Active case counts for each municipality were tracked from the NH and VT state COVID-19 dashboards. We received a total of 283 wastewater samples from all sites during the study period. Viral RNA was detected in 175/283 (61.8%) samples using RT-qPCR and in 195/283 (68.9%) samples using RT-ddPCR. All nine sites showed positivity in the wastewater, with 8/9 (88.8%) sites having over 50% of their samples test positive over the course of the study. Larger municipalities, such as Nashua, Concord, and Lebanon, NH, showed that SARS-CoV-2 positivity in the wastewater can precede spikes in active COVID-19 case counts by as much as 7 days. Smaller municipalities, such as Woodsville, NH and Hartford, VT, showed sporadic SARS-COV-2 detection and did not always precede a rise in active case counts. We detected SARS-CoV-2 RNA in samples from all 9 municipalities tested, including cities and small towns within this region, and showed wastewater positivity as an early indicator of active case count increases in some regions. Some of the smaller rural municipalities with low case counts may require more frequent sampling to detect SARS-CoV-2 in wastewater before a case surge. With timely collection and analysis of wastewater samples, a community could potentially respond to results by increasing public health initiatives, such as tightening mask mandates and banning large indoor gatherings, to mitigate community transmission of SARS-CoV-2. IMPORTANCE Despite vaccination efforts, the delta and omicron variants of SARS-CoV-2 have caused global surges of COVID-19. As the COVID-19 pandemic continues, it is important to find new ways of tracking early signs of SARS-CoV-2 outbreaks. The manuscript outlines how to collect wastewater from treatment facilities, concentrate the virus in a dilute wastewater sample, and detect it using two sensitive PCR-based methods. It also describes important trends in SARS-CoV-2 concentration in wastewater of a rural region of the United States from Fall 2020 - Winter 2021 and demonstrates the utility of wastewater monitoring as a leading indicator of active SARS-CoV-2 cases. Monitoring changes in concentration of SARS-CoV-2 virus in wastewater may offer an early indicator of increased case counts and enable appropriate public health actions to be taken.

Utility of a virtual counselor (VICKY) to collect family health histories among vulnerable patient populations: A randomized controlled trial.

OBJECTIVES: This study is a randomized controlled trial comparing the efficacy of a virtual counselor (VICKY) to the My Family Health Portrait (MFHP) tool for collecting family health history (FHx). METHODS: A total of 279 participants were recruited from a large safety-net hospital and block randomized by health literacy to use one of the digital FHx tools, followed by a genetic counselor interview. A final sample of 273 participants were included for analyses of primary study aims pertaining to tool concordance, which assessed agreement between tool and genetic counselor. RESULTS: Tool completion differed significantly between tools (VICKY = 97%, MFHP = 51%; p < .0001). Concordance between tool and genetic counselor was significantly greater for participants randomized to VICKY compared to MFHP for ascertaining first- and second-degree relatives (ps<.0001), and most health conditions examined. There was significant interaction by health literacy, with greater differences in concordance observed between tools among those with limited literacy. CONCLUSIONS: A virtual counselor overcomes many of the literacy-related barriers to using traditional digital tools and highlights an approach that may be important to consider when collecting health histories from vulnerable populations. PRACTICE IMPLICATIONS: The usability of digital health history tools will have important implications for the quality of the data collected and its downstream clinical utility.

Regulator combinations identify systemic sclerosis patients with more severe disease.

Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder that results in skin fibrosis, autoantibody production, and internal organ dysfunction. We previously identified 4 "intrinsic" subsets of SSc based upon skin gene expression that are found across organ systems. Gene expression regulators that underlie the SSc-intrinsic subsets, or are associated with clinical covariates, have not been systematically characterized. Here, we present a computational framework to calculate the activity scores of gene expression regulators and identify their associations with SSc clinical outcomes. We found that regulator activity scores can reproduce the intrinsic molecular subsets, with distinct sets of regulators identified for inflammatory, fibroproliferative, limited, and normal-like samples. Regulators most highly correlated with modified Rodnan skin score (MRSS) also varied by intrinsic subset. We identified subgroups of patients with fibroproliferative and inflammatory SSc with more severe pathophenotypes, such as higher MRSS and increased likelihood of interstitial lung disease (ILD). Using an independent cohort, we show that the group with more severe ILD was more likely to show forced vital capacity decline over a period of 36-54 months. Our results demonstrate an association among the activation of regulators, gene expression subsets, and clinical variables that can identify patients with SSc with more severe disease.

Mycophenolate Mofetil Treatment of Systemic Sclerosis Reduces Myeloid Cell Numbers and Attenuates the Inflammatory Gene Signature in Skin.

Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.