ESPE Abstracts

Background: Loss-of-function mutations in the Delta Like Non-Canonical Notch Ligand 1 (DLK1) gene have been identified in patients with central precocious puberty (CPP) and were associated with metabolic and reproductive alterations at adulthood.

Objectives: To evaluate the effects of DLK1 loss-of-function mutations on metabolism and other biological process in adult women who had previously CPP in childhood due to DLK1 mutations using whole blood transcriptome.

Patients and Methods: Five female patients with mean age of 32.9 years (26.9 to 39) from two Brazilian families who developed CPP in childhood due to DLK1 mutations were evaluated. Four patients presented overweight/obesity and one healthy weight (IMC: 28.34 ± 2.7 Kg/m2). Whole peripheral blood samples were collected from the five patients and seven female healthy controls, paired by age (26 to 40 years old). RNA extraction was performed using the PAXgene Blood RNA kit. Paired-end library was prepared using the Stranded Total RNA Prep kit, ligation with Ribo-Zero Plus. The RNA sequencing was performed on NextSeq 2000. The normalized counts were obtained with Salmon package. The Principal Component Analysis (PCA) and differential expression genes (DEGs) were undertaken in R software using DESeq2 package. Gene Set Enrichment Analysis (GSEA) was used to evaluate the enriched pathways. The P -value adjusted by false discovery rate was <0.05, and the |log2 foldchange (FC)| was >1.

Results: The PCA demonstrated a clear separation between patients and healthy controls. In the comparison of patients versus healthy controls, among the top upregulated DEGs, we identified upregulation on DEFA4, OLFM4, LCN2, LTF and MMP8 genes, all related to neutrophil activation and previously found to be increased in obese patients. Other upregulated genes were MYL9 and PDGFRB, both associated with platelets activation and risk of injury of vascular remodeling, and TANC1, related to synaptic proteins such as glutamate receptors. The top downregulated DEGs were long non-coding RNAs such as ENSG00000276232 (unknown function) and MAPK8IP1P2, related to short sleep. Interestingly, the miRNA-3648-1 was also downregulated. In line with our findings, the GSEA showed enrichment in pathways of innate and humoral immune response GO biological process terms in patients compared with controls.

Conclusion: An increased expression on neutrophil and platelet activation genes associated with metabolic disorders, such as obesity and diabetes mellitus, and chronic inflammation was identified in adult female patients carrying loss-of-function mutations in DLK1. This study reinforces the link between DLK1 deficiency and metabolic disorders, highlighting the importance of long-term follow-up into adulthood.