Down syndrome, caused in about 95 percent of cases by a full extra copy of chromosome 21, is the most common chromosomal disorder associated with intellectual disability. Its effects ripple across the neurological, musculoskeletal, and cardiovascular systems, and roughly half of all individuals with the condition are born with congenital heart defects. Over the past decade, researchers have documented widespread epigenetic alterations, changes to chemical marks on DNA that regulate gene activity without altering the underlying sequence, in the nuclear DNA of people with trisomy 21. These changes have been linked to features ranging from accelerated aging to hematological and cardiovascular complications. But the mitochondrial genome has remained largely terra incognita for epigeneticists, even as evidence accumulates that mitochondrial dysfunction is a genuine feature of the syndrome.

The reason mitochondria matter so much in Down syndrome comes down to gene dosage. The extra chromosome carries genes involved in oxidative stress pathways, and cells from people with the syndrome show impaired energy metabolism, chronic pro-oxidative states, and deficits in the molecular machinery that produces ATP, the cell’s energy currency. Studies in fibroblasts and brain tissue have repeatedly detected these functional disturbances. Yet measuring mitochondrial function directly is difficult, and one of the most accessible molecular proxies, the number of mitochondrial DNA copies per cell, has yielded only slight and statistically unconvincing increases in prior studies of Down syndrome tissues.

The new study focused on a very specific stretch of the mitochondrial genome: the D-loop, a non-coding regulatory region that contains both the origin of DNA replication and the main transcription start site for the mitochondrial chromosome. The D-loop is where DNA methyltransferases, the enzymes that attach methyl groups to cytosine bases, appear to concentrate their activity on mitochondrial DNA. Although methylation levels across the mitochondrial genome are typically very low, around 0.5 to 1 percent, the D-loop can reach peaks of 10 to 15 percent. Crucially, methylation in this region modulates the binding of TFAM, mitochondrial transcription factor A, a master regulator of both mitochondrial DNA transcription and replication. In diseases from Alzheimer’s to amyotrophic lateral sclerosis to cardiovascular conditions, D-loop methylation has been found altered and sometimes correlated with disease severity and mitochondrial DNA copy number, making it a plausible suspect in Down syndrome as well.

The suspicion was sharpened by a single earlier study from 2011, in which researchers examined immortalized lymphoblastoid cells from six children with Down syndrome and six matched controls. That work identified a breakdown in one-carbon metabolism, the cellular circuit that generates the methyl-donor molecule S-adenosylmethionine, and found reduced levels of this methylating agent inside the mitochondria of Down syndrome cells, along with global hypomethylation of mitochondrial DNA. If mitochondrial methylation capacity was genuinely compromised, it stood to reason that measurable differences should appear in patient blood samples.

To find out, the team led by Andrea Stoccoro and Fabio Coppedè of the University of Pisa, working with colleagues at the University of Rijeka and the Juraj Dobrila University of Pula, recruited 118 participants at the Clinical Hospital Centre Rijeka in collaboration with Down syndrome associations across Croatia. The cohort ranged in age from newborns to 55 years, with every Down syndrome case confirmed by karyotyping and every participant matched by age and sex to a healthy control. Among the newborns, ten had Down syndrome with congenital heart defects, eight had Down syndrome without such defects, and eighteen were healthy controls. The heart defects identified by ultrasound included five atrial septal defects, two atrioventricular septal defects, one ventricular septal defect, one patent ductus arteriosus, and one case of tetralogy of Fallot, and newborn blood samples were drawn within seventy-two hours of birth, before any surgical or medical correction of cardiac malformations could confound the measurements.

Methodologically, the study leaned on methylation-sensitive high-resolution melting, a technique that amplifies a bisulfite-converted segment of DNA and then reads out its methylation level from the melting behavior of the resulting PCR product. The team had previously developed and validated this protocol against pyrosequencing, and in the present study it targeted a 222-base-pair segment of the D-loop containing ten CpG sites. Calibration standards spanning 0 to 100 percent methylation were run in each assay, and an interpolation method developed in the same laboratory converted melting curves into precise single-value methylation percentages. Bisulfite conversion efficiency averaged 99 percent, and equal numbers of patient and control samples were processed together to suppress batch effects. Mitochondrial DNA copy number was measured independently by quantitative PCR comparing a mitochondrial target against the nuclear hemoglobin beta gene, with the caveat that limited DNA availability allowed copy number analysis in only 103 of the 118 participants.

The results were striking in their uniformity. Across individuals, D-loop methylation ranged from 0 to about 8 percent, and mitochondrial DNA copy number ranged from 33 to 900 copies, reflecting enormous person-to-person variability. But the distributions overlapped almost perfectly between groups. Median D-loop methylation was 1.3 percent in controls versus 4.0 percent in the Down syndrome group, a difference that fell short of statistical significance at p equal to 0.28. Mitochondrial DNA copy number medians were similarly indistinguishable, at roughly 128 copies in controls and 149 in the syndrome group, with p equal to 0.22. Age showed no correlation with either biomarker across the entire sample, spanning newborns to mid-adulthood. Sex made no significant difference either, although the authors noted a slight, non-significant tendency toward higher copy number in males. Most importantly for the congenital heart question, comparisons among the three newborn groups, those with Down syndrome and heart defects, those with Down syndrome and healthy hearts, and controls, revealed no differences in either methylation or copy number, with p values of 0.85 and 0.60 respectively.

The authors are careful to frame the work as a pilot study and to spell out its limits. The analysis covered only a small portion of the mitochondrial genome, so alterations elsewhere in the mitochondrial chromosome cannot be excluded. The newborn subgroup, particularly those without heart defects, was small, and too small to permit sex-stratified analysis even though previous genome-wide studies of nuclear DNA in Down syndrome newborns have shown that many methylation differences tied to congenital heart defects are sex-specific. Blood methylation may also simply fail to mirror what happens in fetal cardiac tissue, the site where the relevant biology would actually unfold. Larger cohorts and studies of cardiac tissue itself will be needed before mitochondrial methylation can be definitively ruled out in the syndrome’s cardiovascular complications.

Even so, the null result carries a useful message. It suggests that the mitochondrial dysfunction documented in Down syndrome, including the more pronounced oxidative stress seen in fetuses with congenital heart defects, does not register in the two most accessible mitochondrial molecular readouts available from a routine blood sample. Unlike nuclear DNA methylation, where robust signatures of trisomy 21 have been detected in blood, placenta, buccal cells, brain, and myocardial tissue, the mitochondrial D-loop appears to hold steady. For researchers hunting biomarkers that could distinguish Down syndrome newborns with heart defects from those without, the message is that the mitochondrial D-loop is unlikely to provide one, at least not in peripheral blood. For the broader field of mitochondrial epigenetics, the study adds a well-controlled data point to a growing literature showing that D-loop methylation shifts in some neurological and cardiovascular diseases but not in every condition marked by mitochondrial stress, a reminder that in biology, the absence of a signal can be as informative as its presence.

Subject of Research: Mitochondrial D-loop region methylation and mitochondrial DNA copy number in individuals with Down syndrome

Article Title: Analysis of mitochondrial D-loop region methylation and copy number in peripheral blood DNA of Down syndrome individuals including newborns with and without congenital heart defects

Article References: Analysis of mitochondrial D-loop region methylation and copy number in peripheral blood DNA of Down syndrome individuals including newborns with and without congenital heart defects. (n.d.). https://doi.org/10.1186/s43682-025-00039-x

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00039-x

Keywords: Down syndrome, mitochondrial DNA, D-loop methylation, mtDNA copy number, epigenetics, congenital heart defects, trisomy 21, bisulfite conversion, high-resolution melting, mitochondrial dysfunction, newborns, biomarkers