Rare diseases (RDs) affect millions globally, with over 50% of cases remaining undiagnosed due to limitations in current diagnostic tools. The xPAND project seeks to address these challenges by developing innovative multi-omics approaches that integrate long-read whole-genome sequencing (LR-WGS), RNA-seq, methylation profiling, and the Human Pangenome Reference. Coordinated by Dr Anna Esteve, Functional Genomics Team Leader at CNAG, this comprehensive framework aims to improve diagnostic accuracy, reduce false negatives, and uncover novel molecular mechanisms underlying rare diseases, particularly for underrepresented populations.

The Human Pangenome Reference, built from diverse global populations, and LR-WGS together enable the detection of complex structural variants (e.g., segmental duplications, triplet expansions, mobile element insertions, and pseudogenes), which are often missed by short-read WGS or WES. This approach facilitates the identification of population-specific variants and hidden causal mutations critical for diagnosing rare diseases. Importantly, the project will evaluate customized pangenomes to investigate unsolved RD cases from Latin America, Africa, and Asia, addressing diagnostic inequities and broadening the understanding of global genetic diversity. Integrating these data with RNA-seq and methylation profiling will further elucidate how these variants affect gene expression, splicing, and epigenetic regulation, advancing knowledge of disease mechanisms and diagnostics.

The project also focuses on improving patient-specific RNA defects detection by improving aberrant expression detection methods, achieving higher resolution at exon- and intron-levels to identify subtle expression changes that remain undetected with current gene-level approaches. Additionally, it will study aberrant methylation patterns in promoters and enhancer regions. These advancements will facilitate the reclassification of variants of uncertain significance (VUS) and the prioritization of novel gene candidates for further investigation.

In the context of X-linked disorders, the xPAND project addresses the underdiagnosis of females investigating non-random X-chromosome inactivation (NR-XCI). Current clinical tests, such as the androgen receptor methylation assay, are limited by their locus-centric nature, lack of haplotype resolution, and inability to determine which allele is preferentially expressed or silenced. By integrating phased RNA-seq and methylome data, xPAND will investigate NR-XCI as a complementary diagnostic tool, shedding light on phenotypic variability and incomplete penetrance in female carriers. Furthermore, studying tissue-specific NR-XCI patterns will clarify cases where blood-based assessments are insufficient, improving diagnostic precision for X-linked disorders in females.

Through these advancements, the xPAND project aligns with the objectives of the Diagnostic, Prognostic, and Therapeutic Tools (DPT) subarea, delivering scalable pipelines suitable for clinical implementation. By investigating unsolved RD cases from underrepresented regions and addressing critical diagnostic gaps in rare disease genomics, xPAND aims to reduce the diagnostic odyssey, improve equity in diagnostics, and advance precision medicine on a global scale.

The project has been funded by the Ministry of Science, Innovation and Universities, the State Research Agency, and the European Regional Development Fund (ERDF).