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Genetic Variability and Trajectories of DNA Methylation May Support a Role for HAMP in Patient Outcomes After Aneurysmal Subarachnoid Hemorrhage

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Abstract

Background/Objective

Preclinical evidence suggests that iron homeostasis is an important biological mechanism following aneurysmal subarachnoid hemorrhage (aSAH); however, this concept is underexplored in humans. This study examined the relationship between patient outcomes following aSAH and genetic variants and DNA methylation in the hepcidin gene (HAMP), a key regulator of iron homeostasis.

Methods

In this exploratory, longitudinal observational study, participants with verified aSAH were monitored for acute outcomes including cerebral vasospasm (CV) and delayed cerebral ischemia (DCI) and evaluated post-discharge at 3 and 12 months for long-term outcomes of death and functional status using the Modified Rankin Scale (mRS; poor = 3–6) and Glasgow Outcome Scale (GOS; poor = 1–3). Participants were genotyped for two genetic variants, and DNA methylation data were collected from serial cerebrospinal fluid over 14 days post-aSAH at eight methylation sites within HAMP. Participants were grouped based on their site-specific DNA methylation trajectory, with and without correcting for cell-type heterogeneity (CTH), and the associations between genetic variants and inferred DNA methylation trajectory groups and patient outcomes were tested. To correct for multiple testing, an empirical significance threshold was computed using permutation testing.

Results

Genotype data for rs10421768 and rs7251432 were available for 241 and 371 participants, respectively, and serial DNA methylation data were available for 260 participants. Acute outcome prevalence included CV in 45% and DCI in 37.1% of the overall sample. Long-term outcome prevalence at 3 and 12 months included poor GOS in 23% and 21%, poor mRS in 31.6% and 27.3%, and mortality in 15.1% and 18.2%, respectively, in the overall sample. Being homozygous for the rs7251432 variant allele was significantly associated with death at 3 months (p = 0.003) and was the only association identified that passed adjustment for multiple testing mentioned above. Suggestive associations (defined as trending toward significance, p value < 0.05, but not meeting empirical significance thresholds) were identified between the homozygous variant allele for rs7251432 and poor GOS and mRS at 3 months (both p = 0.04) and death at 12 months (p = 0.02). For methylation trajectory groups, no associations remained significant after correction for multiple testing. However, for methylation trajectory groups not adjusted for CTH, suggestive associations were identified between cg18149657 and poor GOS and mRS at 3 months (p = 0.003 and p = 0.04, respectively) and death at 3 months (p = 0.04), and between cg26283059 and DCI (p = 0.01). For methylation trajectory groups adjusted for CTH, suggestive associations were identified between cg02131995 and good mRS at 12 months (p = 0.02), and between cg26283059 and DCI (p = 0.01).

Conclusions

This exploratory pilot study offers preliminary evidence that HAMP may play a role in patient outcomes after aSAH. Replication of this study and mechanistic investigation of the role of HAMP in patient outcomes after aSAH are needed.

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Acknowledgements

We would also like to acknowledge the anonymous reviewers who took the time to critically evaluate this paper. Their detailed feedback greatly improved the clarity and quality of this work for the scientific community.

Funding

Research reported in this publication was supported by the National Instiute of Nursing Research of the National Institutes of Health under Award Numbers F31NR017311, R01NR004339, R01NR013610, and T32NR009759 and with additional support from the International Society of Nurses in Genetics, University of Pittsburgh Leslie A. Hoffman Endowed Research Award, Nightingale Awards of Pennsylvania, and Eta Chapter, Sigma Theta Tau, Inc. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or supporting foundations.

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Authors and Affiliations

  1. Department of Health Promotion and Development, School of Nursing, University of Pittsburgh, 440 Victoria Building, 3500 Victoria Street, Pittsburgh, PA, 15261, USA

    Lacey W. Heinsberg & Yvette P. Conley

  2. Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

    Annie I. Arockiaraj, John R. Shaffer, Daniel E. Weeks & Yvette P. Conley

  3. Department of Acute and Tertiary Care, School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA

    Elizabeth A. Crago & Paula R. Sherwood

  4. Department of Health and Community Systems, School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA

    Dianxu Ren & Susan M. Sereika

  5. Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    John R. Shaffer

  6. Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

    Daniel E. Weeks

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  1. Lacey W. Heinsberg
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Contributions

LWH contributed to the study conception and design, analysis and interpretation of data, and drafted, critically revised, and gave final approval for the manuscript. AIA contributed to the analysis of data and critically revised and gave final approval for the manuscript. EAC contributed to the acquisition and interpretation of data and critically revised and gave final approval for the manuscript. DR contributed to the analysis and interpretation of data and critically revised and gave final approval for the manuscript. JRS contributed to analysis and interpretation of the data and critically revised and gave final approval for the manuscript. PRS contributed to the acquisition of data and critically revised and gave final approval for the manuscript. SMS contributed to the analysis and interpretation of data and critically revised and gave final approval for the manuscript. DEW contributed to the study design, analysis and interpretation of data, and critically revised and gave final approval for the manuscript. YPC contributed to the study conception and design, interpretation of data, and critically revised and gave final approval for the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors meet authorship criteria, have read and approved the published work, and certify that they have participated sufficiently in the work to take responsibility for the content including the concept, design, analysis, writing, or revision (see author contributions above).

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Correspondence to Lacey W. Heinsberg.

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Conflict of interest

LW Heinsberg reports grants from the National Institute of Nursing Research, University of Pittsburgh, Eta Chapter, Sigma Theta Tau, Inc., and International Society of Nurses in Genetics during the conduct of this study. YP Conley and PR Sherwood reports grants from the National Institute of Nursing Research. EA Crago reports grants from the University of Pittsburgh. The remaining authors report no conflicts of interest to disclose.

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Institutional review board approval at the University of Pittsburgh has been obtained (IRB Approval Number 021039), and we have adhered to ethical considerations in the protection of all human subjects involved.

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Heinsberg, L.W., Arockiaraj, A.I., Crago, E.A. et al. Genetic Variability and Trajectories of DNA Methylation May Support a Role for HAMP in Patient Outcomes After Aneurysmal Subarachnoid Hemorrhage. Neurocrit Care 32, 550–563 (2020). https://doi.org/10.1007/s12028-019-00787-4

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