The team, led by Sampreeti Tahbildar and Ashis Das of the Molecular Parasitology and Systems Biology Lab at BITS Pilani, published their findings in Frontiers in Malaria on 11 November 2025. Their approach relied on array-based comparative genomic hybridization, a technique that measures the relative abundance of every segment of a parasite genome by competitively hybridizing fluorescently labeled test and reference DNA onto a dense microarray. Rather than sequencing whole genomes, which can struggle with the repetitive, GC-rich regions that litter malaria parasite chromosomes, the researchers designed a custom 2 × 400K tiling array carrying 418,577 oligonucleotide probes spaced an average of just 56 base pairs apart across the entire P. vivax genome. That density, anchored to the P. vivax Sal-1 reference genome, allowed them to detect copy number changes as small as 100 base pairs and as large as nearly 1.5 megabases.

The clinical material came from patients admitted to S.P. Medical College in Bikaner, in northwestern India, a region where P. vivax remains endemic. Blood samples were drawn after informed consent, processed within fifteen minutes of collection, and confirmed as pure P. vivax infections using microscopy, rapid diagnostic tests, and an 18S rRNA gene-based multiplex PCR that ruled out P. falciparum co-infection. Three isolates came from patients with cerebral malaria, defined by a Glasgow coma scale score below 11, and three came from patients with uncomplicated disease. Genomic DNA from each group was pooled, labeled with distinct fluorescent dyes, and hybridized against each other on the tiling array. CNVs were called using the ADM-2 algorithm, a standard tool in Agilent array-CGH analysis, with stringent filters requiring at least three consecutive probes showing an average absolute log2 ratio of 0.3 or greater, a threshold designed to eliminate single-probe artifacts arising from sequence mismatches.

The results were striking in their breadth. Copy number variations appeared across all 14 chromosomes of the P. vivax genome, affecting a total of 2,138 genes. Altered intervals ranged from roughly 100 base pairs to approximately 1,429 kilobases. The single most extensive CNV spanned about 1,450 kilobases on chromosome 12, covering 316 genes in a continuous stretch of amplification. Chromosome 14 and chromosome 10 also carried large amplified blocks, encompassing 100 and 96 genes respectively. Many of the affected genes encode hypothetical proteins whose functions remain uncharacterized, a reflection of the persistent difficulty in maintaining P. vivax in continuous laboratory culture, which has hampered functional studies of the parasite for decades.

Among the most consequential findings were CNVs in genes linked to antimalarial drug resistance. The team detected amplifications in three genes encoding putative multidrug resistance proteins belonging to ABC transporter families, including PVX_097025, PVX_080100, and PVX_118100. Amplification of the P. falciparum ortholog of PVX_080100 has previously been associated with mefloquine resistance, and increased copy number of the multidrug resistance gene 1, mdr1, has been repeatedly linked to elevated drug tolerance in both P. falciparum and P. vivax. Earlier work by Suwanarusk and colleagues showed that pvmdr1 amplification in Thai P. vivax isolates correlated with higher IC50 values for mefloquine and artesunate, providing functional evidence that copy number changes at this locus translate into measurable drug resistance phenotypes. The current study also identified amplification of a putative chloroquine resistance marker protein on chromosome 12 and deletion of a putative chloroquine resistance transporter on chromosome 1, echoing field observations from the Brazilian Amazon where multi-copy pvcrt-o has been tied to chloroquine treatment failure.

Equally significant were the CNVs detected in genes encoding surface antigens, the molecular interface between parasite and host immune system. All 24 tryptophan-rich antigens, or TRAgs, showing copy number changes were amplified, consistent with the tendency of this gene family to proliferate through duplication and diversification. One amplified TRAg, encoded by PVX_088850, is known to interact with PvMSP7 at the merozoite surface, suggesting a role in stabilizing protein complexes required for red blood cell invasion. The study also found copy number changes in 23 genes encoding serine-repeat antigens, all clustered on chromosome 4, with 19 in deleted regions and four in amplified regions. Amplifications were detected in genes for reticulocyte-binding proteins, or PvRBPs, which mediate invasion of young red blood cells, and in genes for rhoptry proteins, early transcribed membrane proteins, and cytoadherence-linked asexual proteins located on chromosomes 7 and 14. In P. falciparum, CLAG proteins are known to drive cytoadherence, the process by which infected erythrocytes bind to host endothelial cells, and to form nutrient uptake channels at the host cell membrane.

Perhaps the most intriguing signal came from the vir gene family, a large subtelomeric multigene superfamily unique to P. vivax. The analysis identified CNVs in 117 unique vir genes, with 84 located in amplified regions, 10 in deleted regions, and 23 showing both gains and losses. Vir genes have been proposed to mediate cytoadherence of infected red blood cells to host endothelial cells, a process that can obstruct microvasculature and contribute to tissue damage, and their extensive sequence diversity is thought to facilitate antigenic variation, allowing the parasite to stay one step ahead of host immunity. Amplification of vir genes in cerebral malaria isolates could, in principle, enhance both immune evasion and the adhesive phenotypes implicated in severe disease, though the authors are careful to note that this remains a hypothesis requiring functional validation.

Beyond coding regions, the study detected CNVs in intergenic stretches of the P. vivax genome, a finding that may carry regulatory implications. Intergenic regions harbor promoters, enhancers, and insulators that govern the expression of neighboring genes, and copy number changes in these non-coding sequences can alter the accessibility of regulatory elements to transcription factors or disrupt long-range chromatin interactions. In P. falciparum, intergenic regions are known to give rise to regulatory long non-coding RNAs that influence gene expression, sexual development, and parasite transmission. In humans, non-coding CNVs have been shown to modify gene regulation and phenotypic outcomes. By analogy, intergenic CNVs in P. vivax could reshape the regulatory landscape of genes encoding surface antigens, drug resistance proteins, and stress-related factors, though the authors emphasize that experimental studies are needed to confirm any such role.

The study also documented copy number changes in genes encoding components of the ubiquitin-proteasome system, with 20 genes from the ubiquitin machinery and 9 from the proteasomal degradation pathway showing CNVs. The proteasome serves as the primary engine of protein degradation in Plasmodium, and ubiquitylation levels rise during the transition from intracellular schizont to extracellular merozoite stages. Ubiquitin modification has also been associated with altered parasite susceptibility to multiple antimalarials, making these CNVs potentially relevant to treatment outcomes. Additional amplifications were detected in 12 genes encoding PHIST proteins, which in P. falciparum participate in host cell remodeling and have been differentially expressed in cerebral and pregnancy-associated malaria, as well as in approximately 80 genes encoding ribosomal complex proteins and in the gene for plasmepsin IV, an aspartic protease involved in hemoglobin digestion within the parasite food vacuole.

The authors are candid about the limitations of their work. All samples came from a single location, Bikaner, and were collected between 2007 and 2008, which may constrain the generalizability of the observed CNV patterns. The small sample size, three cerebral malaria isolates and three uncomplicated controls, reflects the practical difficulty of recruiting severe P. vivax cases, which remain comparatively rare relative to uncomplicated infections. The pooled design provides a group-level overview rather than individual-level resolution, and the authors explicitly state that they cannot conclude whether the observed CNVs contribute directly to disease severity. What they can say is that these copy number changes were present in cerebral malaria isolates when compared with uncomplicated cases, and that they cluster in genes and regions with plausible roles in immune evasion, drug resistance, cytoadherence, and host-pathogen interaction. To their knowledge, no previous whole-genome CNV analysis has been performed on P. vivax cerebral malaria isolates, making this a foundational dataset for future investigations. The microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE288219, allowing other groups to interrogate the findings independently. As P. vivax continues to shed its reputation as a benign parasite, studies like this one point toward a future where genomic surveillance of copy number variation could identify biomarkers for severe disease and inform more effective strategies for malaria control and treatment.

Subject of Research: Copy number variation analysis of Plasmodium vivax isolates from cerebral and uncomplicated malaria cases in India

Article Title: Genomic dynamics of clinical Plasmodium vivax: comparative genomic hybridization in severe malaria cases

Article References: Tahbildar, S., Boopathi, P. A., Kochar, S. K., Kochar, D. K., Aiyaz, M., Mugasimangalam, R. C., Rao, S. N., & Das, A. (2025). Genomic dynamics of clinical Plasmodium vivax: comparative genomic hybridization in severe malaria cases. Frontiers in Malaria, 3, Article 1667330. https://doi.org/10.3389/fmala.2025.1667330

Image Credits: AI Generated

DOI: 10.3389/fmala.2025.1667330

Keywords: Plasmodium vivax, copy number variation, cerebral malaria, comparative genomic hybridization, drug resistance, surface antigens, vir genes, intergenic regions, India, malaria severity, microarray, genomics

The scientific backdrop to this study is a genuine epidemiological puzzle. For decades, researchers have known that women who have given birth to more children experience a lower incidence of breast cancer after menopause, with the risk declining roughly linearly as parity increases. Pregnancy induces profound and lasting changes in breast tissue, including differentiation of the mammary epithelium, and many investigators have assumed that these changes would be reflected in altered gene expression profiles that could be detected decades later. Yet the precise molecular mechanism by which childbearing confers its protective effect has remained elusive, and attempts to pin it down at the level of tissue transcriptomics have produced inconsistent results.

The NOWAC research group, led by Eiliv Lund of UiT The Arctic University of Norway in Tromsø, had previously reported a striking observation that seemed to point toward a mechanism. In studies of peripheral blood cells from healthy postmenopausal women, the team demonstrated that increasing parity was associated with a linear decrease in the expression of hundreds of genes. This gene expression signature in blood cells appeared to mirror the linear decline in postmenopausal breast cancer incidence with increasing parity. Intriguingly, however, the same parity-related expression changes were not observed in the blood cells of women who had already developed breast cancer, suggesting that the signature might represent a protective state that is lost or overridden during carcinogenesis.

Those earlier findings raised an obvious and important question: if parity reshapes gene expression in blood cells in a way that tracks cancer risk, does it also reshape gene expression in the breast tissue itself, where tumors actually arise? Answering this question required tissue samples that are extraordinarily difficult to obtain from healthy women, because there is ordinarily no clinical reason to biopsy normal breast tissue. The NOWAC infrastructure, a prospective cohort study that enrolled 172,000 women between 1991 and 2007, provided the framework needed to attempt it.

Between 2006 and 2010, the researchers recruited women in two ways. Women enrolled in NOWAC who received a diagnostic biopsy for breast cancer were asked to provide a second biopsy specifically for research purposes, with the collaboration of surgeons at ten hospitals participating in the Norwegian Breast Cancer Group. In parallel, normal breast tissue was collected from NOWAC participants undergoing the national mammographic screening program, an effort made possible by a feasibility study showing that 74 percent of healthy women approached at a screening center in Tromsø agreed to donate tissue. The final analytical dataset consisted of 279 age-matched case-control pairs, and the paired matched design was maintained throughout all laboratory analyses, a methodological choice that strengthens the comparison between cancer patients and healthy controls.

The laboratory work relied on Illumina microarray technology to measure genome-wide gene expression, with differential expression analysis performed using the limma Bioconductor package in R, a standard and widely trusted tool for microarray statistics. To characterize the molecular subtype of each tumor and each normal tissue sample, the researchers applied the PAM50 test, a 50-gene classifier that assigns breast tumors to intrinsic subtypes such as luminal A, luminal B, HER2-enriched, and basal-like. The statistical framework controlled the false discovery rate at a q-value threshold of 0.05, ensuring that reported findings were unlikely to be artifacts of multiple testing across thousands of genes.

The results on the cancer side were dramatic, but not in the way the parity hypothesis would predict. When the researchers compared breast cancer tissue with normal breast tissue, they found that nearly the entire transcriptome was differentially expressed: 10,013 of the 11,308 genes assessed showed statistically significant differences between cases and controls. Of these, 5,768 genes were upregulated and 4,245 genes were downregulated in cancer tissue, a sweeping molecular signature of the malignant state that confirms the biological gulf between tumor and normal tissue. This massive case-control signal also demonstrates that the study had ample statistical power to detect expression differences, which makes the central null finding all the more meaningful.

That central finding is the study’s headline result. When parity, classified into three categories of zero children, one to three children, and four to eight children, was incorporated into the statistical model as an explanatory variable, only three genes in breast cancer tissue showed significant changes associated with parity, and not a single gene in normal breast tissue did. Gene set enrichment analyses, which test whether predefined biological pathways are collectively altered, returned non-informative results. Furthermore, the PAM50 classification failed to establish any relationship between parity and the distribution of intrinsic molecular subtypes in either tissue type. In other words, across the entire measurable transcriptome, the number of full-term pregnancies left virtually no trace in the breast.

The authors conclude that their study revealed no significant differences in gene expression between normal and breast cancer tissue in relation to the number of full-term pregnancies. This is a genuine null result from a well-powered, carefully matched design, and null results of this kind carry real scientific weight. They effectively rule out a whole class of mechanistic explanations: whatever protects parous women from postmenopausal breast cancer, it does not appear to operate through durable, detectable changes in the steady-state expression levels of genes in breast tissue, whether healthy or malignant. The protective signal that the team previously observed in peripheral blood cells now stands in sharper contrast, suggesting that the parity-related biology may be systemic rather than resident in the breast itself, or that it operates through mechanisms invisible to bulk transcriptomics, such as epigenetic marks, stromal architecture, immune surveillance, or transient changes that fade long before menopause.

The study also stands as a testament to the value of long-term prospective cohorts with biobanks. Only a resource like NOWAC, which links decades of lifestyle and reproductive history data to consented tissue donations and national cancer registry records, could assemble 279 matched pairs of cancer and normal breast tissue with complete parity information. The work was supported by the European Research Council, the Northern Norway Regional Health Authority, and the Norwegian Cancer Society, and the authors emphasize that the funders played no role in the design, analysis, or interpretation of the study. For researchers hunting the molecular basis of pregnancy’s protective effect, the message is clear: the answer will not be found by simply cataloging which genes are switched on or off in breast tissue. The search must now move to other layers of biology, and this study, by closing one door definitively, helps point the way.

Subject of Research: The effect of parity on global gene expression in normal breast tissue and breast cancer tissue

Article Title: No impact of parity on global gene expression levels in breast cancer tissue and normal breast tissue – a nested case-control study in the Norwegian Women and Cancer study

Article References: Lund, E., Krum-Hansen, S., Olsen, K. S., Shvetsov, N., Snapkow, I., Gavriluk, O., Busund, L.-T. R., Frantzen, J. O., Holden, M., & Holden, L. (2026). No impact of parity on global gene expression levels in breast cancer tissue and normal breast tissue – a nested case-control study in the Norwegian Women and Cancer study. BMC Cancer. https://doi.org/10.1186/s12885-026-17044-5

Image Credits: AI Generated

DOI: 10.1186/s12885-026-17044-5

Keywords: breast cancer, parity, gene expression, NOWAC study, transcriptomics, microarray, PAM50, nested case-control, epidemiology, reproductive history, cancer prevention, Norway