Identification of cell-free DNA methylation patterns unique to the human left ventricle as a potential indicator of acute cellular rejection.

Increased levels of donor-derived cell-free DNA (dd-cfDNA) in recipient plasma have been associated with rejection after transplantation. DNA sequence differences have been used to distinguish between donor and recipient, but epigenetic differences could also potentially identify dd-cfDNA. This pilot study aimed to identify ventricle-specific differentially methylated regions of DNA (DMRs) that could be detected in cfDNA. We identified 24 ventricle-specific DMRs and chose two for further study, one on chromosome 9 and one on chromosome 12. The specificity of both DMRs for the left ventricle was confirmed using genomic DNA from multiple human tissues. Serial matched samples of myocardium (n = 33) and plasma (n = 24) were collected from stable adult heart transplant recipients undergoing routine endomyocardial biopsy for rejection surveillance. Plasma DMR levels increased with biopsy-proven rejection grade for individual patients. Mean cellular apoptosis in biopsy samples increased significantly with rejection severity (2.4%, 4.4% and 10.0% for ACR 0R, 1R, and 2R, respectively) but did not show a consistent relationship with DMR levels. We identified multiple DNA methylation patterns unique to the human ventricle and conclude that epigenetic differences in cfDNA populations represent a promising alternative strategy for the non-invasive detection of rejection.

Aorta-specific DNA methylation patterns in cell-free DNA from patients with bicuspid aortic valve-associated aortopathy.

BACKGROUND: The dilation of the aorta that occurs as a consequence of a congenitally bicuspid aortic valve (BAV) is associated with a risk of dissection, aneurysm or rupture. With progressive aortopathy, surgery is often recommended, but current patient selection strategies have limitations. A blood-based assay to identify those who would most benefit from prophylactic surgery would be an important medical advance. In a proof-of-concept study, we sought to identify aorta-specific differentially methylated regions (DMRs) detectable in plasma cell-free DNA (cfDNA) obtained from patients undergoing surgery for BAV-associated aortopathy. METHODS: We used bioinformatics and publicly available human methylomes to identify aorta-specific DMRs. We used data from 4D-flow cardiac magnetic resonance imaging to identify regions of elevated aortic wall shear stress (WSS) in patients with BAV-associated aortopathy undergoing surgery and correlated WSS regions with aortic tissue cell death assessed using TUNEL staining. Cell-free DNA was isolated from patient plasma, and levels of candidate DMRs were correlated with aortic diameter and aortic wall cell death. RESULTS: Aortic wall cell death was not associated with maximal aortic diameter but was significantly associated with elevated WSS. We identified 24 candidate aorta-specific DMRs and selected 4 for further study. A DMR on chromosome 11 was specific for the aorta and correlated significantly with aortic wall cell death. Plasma levels of total and aorta-specific cfDNA did not correlate with aortic diameter. CONCLUSIONS: In a cohort of patients undergoing surgery for BAV-associated aortopathy, elevated WSS created by abnormal flow hemodynamics was associated with increased aortic wall cell death which supports the use of aorta-specific cfDNA as a potential tool to identify aortopathy and stratify patient risk.

Preterm Infant Growth Velocity Calculations: A Systematic Review.

CONTEXT: Clinicians assess the growth of preterm infants and compare growth velocity using a variety of methods. OBJECTIVE: We determined the numerical methods used to describe weight, length, and head circumference growth velocity in preterm infants; these methods include grams/kilogram/day (g/kg/d), grams/day (g/d), centimeters/week (cm/week), and change in z scores. DATA SOURCES: A search was conducted in April 2015 of the Medline database by using PubMed for studies that measured growth as a main outcome in preterm neonates between birth and hospital discharge and/or 40 weeks' postmenstrual age. English, French, German, and Spanish articles were included. The systematic review was conducted by using Preferred Reporting Items for Systematic Reviews and Meta-analyses methods. STUDY SELECTION: Of 1543 located studies, 373 (24%) calculated growth velocity. DATA EXTRACTION: We conducted detailed extraction of the 151 studies that reported g/kg/d weight gain velocity. RESULTS: A variety of methods were used. The most frequently used method to calculate weight gain velocity reported in the 1543 studies was g/kg/d (40%), followed by g/d (32%); 29% reported change in z score relative to an intrauterine or growth chart. In the g/kg/d studies, 39% began g/kg/d calculations at birth/admission, 20% at the start of the study, 10% at full feedings, and 7% after birth weight regained. The kilogram denominator was not reported for 62%. Of the studies that did report the denominators, the majority used an average of the start and end weights as the denominator (36%) followed by exponential methods (23%); less frequently used denominators included birth weight (10%) and an early weight that was not birth weight (16%). Nineteen percent (67 of 355 studies) made conclusions regarding extrauterine growth restriction or postnatal growth failure. Temporal trends in head circumference growth and length gain changed from predominantly cm/wk to predominantly z scores. LIMITATIONS AND CONCLUSIONS: The lack of standardization of methods used to calculate preterm infant growth velocity makes comparisons between studies difficult and presents an obstacle to using research results to guide clinical practice.