Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y chromosome segment lengths in phenotypic males with 46,XX testicular disorder/difference of sex development

Berglund A; Johannsen EB; Skakkebaek A; Chang S; Rohayem J; Laurentino S; Horlyck A; Drue SO; Bak EN; Fedder J; Tuttelmann F; Gromoll J; ust J; Gravholt CH

doi:10.1530/ey.22.14.6

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ESPE Yearbook of Paediatric Endocrinology (2025) 22 14.6 | DOI: 10.1530/ey.22.14.6

ESPEYB25 14. The Year in Science and Medicine Genomics – Long-Read Sequencing (7 abstracts)

14.6. Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y chromosome segment lengths in phenotypic males with 46,XX testicular disorder/difference of sex development

Berglund A , Johannsen EB , Skakkebæk A , Chang S , Rohayem J , Laurentino S , Hørlyck A , Drue SO , Bak EN , Fedder J , Tüttelmann F , Gromoll J , Just J & Gravholt CH

Biol Sex Differ. 2024 Oct 8;15(1):77. https://doi.org/10.1186/s13293-024-00654-8

Brief Summary: This integrative omics study investigated 11 individuals diagnosed with 46,XX testicular disorder/difference of sex development (DSD). By combining long-read sequencing, RNA-seq, and DNA methylation analyses, the authors identified four structural subtypes of Y chromosome translocations and demonstrated their association with distinct molecular and clinical phenotypes.

In rare sex development disorders such as 46,XX testicular DSD, structural genomic complexity often remains undetected by conventional diagnostic methods. The authors addressed this challenge using long-read sequencing technology, which enabled high-resolution mapping of Y chromosome breakpoints and subclassification into SRY-negative, short, medium, and long Yp arm subgroups.

The study identified variable deletions in the PRKY, AMELY, and TBLY1 genes, and confirmed that most translocated PAR1 segments were of Y-chromosomal origin. Integration of epigenetic and transcriptomic data revealed that the length of the Y segment influenced not only Y-linked gene expression but also regulatory patterns on autosomal and X-linked genes. These molecular alterations were associated with clinical features such as short stature, reduced lean body mass, and small testicular volume.

These data strongly highlight the diagnostic and subclassification value of long-read sequencing in rare DSD cases. Although direct functional experiments (e.g., in vitro assays or protein-level studies) were not performed, the integration of genomic, transcriptomic, and epigenetic data revealed biologically meaningful patterns. This multi-omics approach sets a new standard for the molecular characterization of complex DSD phenotypes and facilitates more individualized clinical management.