Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study

Our plain-English summary of Next Generation Sequencing for miRNA Detection on the Exhaled Breath Condensate: A Pilot Study, by R. Cherchi, R. Cusano, S. Orrù, et al (Epigenetics Insights, 2023).

The study

• Authors: R. Cherchi, R. Cusano, S. Orrù, P.A. Ferrari, M. Massidda, G. Fotia, S. De Matteis, P. Cocco
• Journal: Epigenetics Insights
• Published: 2023;16:25168657231160985
• Cited by: 17 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Introduction: Exhaled breath condensate (EBC) sampling has been suggested as a less-invasive and cost-effective method to detect biological macromolecules, including miRNA. To explore the feasibility of its use as a biomarker of early effects of asbestos exposure, we conducted a preliminary test on male volunteers by comparing the miRNA profile in the EBC and the plasma using 2 different sequencing platforms.

Methods: Six male volunteers, all retired and unexposed to dust or fumes, participated in the test. RNA was extracted from 200 μL EBC samples and same-size plasma samples. Sample aliquots were processed in 2 laboratories using 2 different sequencing platforms: a MiSeq Illumina® platform and a more performing HiSeq Illumina® platform.

Results: The HiSeq3000® sequencing platform identified twice as many unique molecular indexes (UMI)-validated miRNA as the MiSeq® platform. The Spearman's correlation coefficient between EBC counts and plasma counts was significant in 5/6 subjects with either platform (MiSeq® = 0.128-0.508, P = .026-<.001; HiSeq® = 0.156-0.412, P = .001-<.001). The intraclass correlation coefficient confirmed the consistency of the miRNA profile over the 6 participants with both biospecimens. Exploring the agreement between the EBC and plasma samples with Bland-Altman plots showed that using the HiSeq3000® platform substantially improved the EBC miRNA detection rate.

Conclusion: Our preliminary study confirms that, when using the HiSeq® sequencing platform, EBC sampling is a suitable, non-invasive method to detect the miRNA profile in healthy subjects.

© The Author(s) 2023.

In plain English

This pilot study from Sardinia, Italy, tested whether microRNA could be measured in exhaled breath condensate. MicroRNAs are small RNA molecules studied as possible early disease markers; breath condensate is moisture collected by breathing into a cooled device. The work was a first step toward a planned project in people formerly exposed to asbestos. The six participants were retired men aged 65 to 73 without respiratory disease. An occupational physician judged from their work histories that they had never been exposed to respiratory hazards. Breath and blood plasma samples were analysed on two sequencing machines. The HiSeq3000 machine identified 600 validated microRNAs, against 303 with the MiSeq machine. Breath and plasma counts were statistically significantly correlated, at fair to moderate levels, in five of the six men on each machine. With the HiSeq3000, 239 microRNAs were found in all six men. It also detected 33 of 42 microRNAs that earlier studies had reported as altered in malignant mesothelioma patients. The authors concluded that measuring microRNA in breath condensate is feasible, and that more research is needed on how closely it matches plasma. They named the small number of participants as a limitation.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Pilot (preliminary) laboratory feasibility study comparing breath condensate with plasma, and two sequencing platforms, in healthy volunteers
• Who was studied: Six retired male volunteers aged 65 to 73, without malignant or non-malignant respiratory disease, in Cagliari, Sardinia, Italy (ethics approval 1 October 2021; sampling dates not given)
• Exposure: Participants were not an asbestos-exposed group: an occupational physician judged from their work histories that they had never been exposed to respiratory hazards. What was compared was the sample type (exhaled breath condensate vs plasma) and the sequencing platform (MiSeq vs HiSeq3000).
• Main results:
  • The HiSeq3000 platform identified 600 UMI-validated miRNA; the MiSeq platform identified 303.
  • Per-person Spearman correlations between breath condensate and plasma miRNA counts were fair to moderate and were statistically significant in all but 1 subject on each platform. Not significant were subject 2 on MiSeq (.103, P = .074) and subject 3 on HiSeq3000 (.070, P = .089).
  • Across the 6 participants, the total number of UMI-validated miRNA reads in breath condensate was not correlated with that in plasma on either platform.
  • Agreement across participants (intraclass correlation) with HiSeq3000 absolute counts was 0.9114 (95% CI 0.9009-0.9213) for plasma, rated excellent, and 0.5700 (0.5359-0.6043) for breath condensate, rated fair. With MiSeq absolute counts it was 0.8371 (0.8117-0.8608) for breath condensate and 0.6703 (0.6281-0.7116) for plasma.
  • The authors interpreted the Bland-Altman plots as showing insufficient miRNA detection in breath condensate with MiSeq, and an improvement with HiSeq3000.
  • With HiSeq3000, 239 miRNA (39.8%) were detected in all subjects; with MiSeq, 10 (3.3%) were.
  • Of 42 miRNA that earlier papers reported as altered in malignant mesothelioma patients, this study detected 28 with MiSeq and 33 with HiSeq. With HiSeq, 16 of these were found in both the breath condensate and the plasma of all participants.
• The authors' conclusion: The authors concluded that monitoring the miRNA profile in exhaled breath condensate is feasible. They added that further investigation is needed to confirm how far it corresponds to the plasma profile, and they recommended high-throughput sequencing.
• Limitations the authors state:
  • The small number of participants limits interpretation of between-person variation and of the correlation between breath condensate and plasma.
  • The small study size did not allow the authors to explore whether age or gender modify the miRNA profile.

Topics

Medical terms in this study, explained in our Lexicon: Biomarker, Asbestos Exposure.

Related studies

• Increased alveolar nitric oxide concentration and high levels of leukotriene B(4) and 8-isoprostane in exhaled breath condensate in patients with asbestosis — H. Lehtonen, P. Oksa, et al. (2007)
• Increased 8-isoprostane, a marker of oxidative stress in exhaled breath condensate in subjects with asbestos exposure — D. Pelclová, Z. Fenclová, et al. (2008)
• Use of exhaled breath condensate to investigate occupational lung diseases — M. Corradi, P. Gergelova, et al. (2010)
• Oxidative stress markers in exhaled breath condensate in lung fibroses are not significantly affected by systemic diseases — D. Pelclová, Z. Fenclová, et al. (2011)
• Leukotrienes B4, C4, D4 and E4 in the exhaled breath condensate (EBC), blood and urine in patients with pneumoconiosis — D. Pelclová, Z. Fenclová, et al. (2012)
• Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study — M. Lo Iacono, V. Monica, et al. (2015)

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