Next generation sequencing for the detection of hypopituitarism candidate genes using molecular inverted probes in Argentinean children

VISHNOPOLSKA SA; MORTENSEN AH; KESELMAN A; CAMPER SA; BUSTAMANTE JP; BERGADA I; LEMONS R; MARÍA INÉS PÉREZ MILLÁN; MARTI MA; BRASLAVSKY D; KITZMAN JO

Congreso; The Endocrine Society´s Annual Meeting; 2017

Congenital combined pituitary hormone deficiency (CPHD) arises from defects in pituitary development and is sometimes associated with craniofacial abnormalities. Our objectives are to understand this disease pathophysiology and to improve molecular diagnosis and treatment. Mutations in the transcription factor PROP1 are the most common known genetic cause of the disorder. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. This study established new mechanisms underlying PROP1?s role in pituitary progenitor cell regulation and offers new candidate genes for CPHD that remain unexplained. The overwhelming majority of patients do not have identified mutations in any of these genes. Our hypothesis is that the genetic factors that cause hormone deficiency are oligogenic and represent a collection of genes expressed in the developing embryonic pituitary, midline, and hypothalamus. We set up a new approach based on Molecular inversion probe (MIP) capture. In this assay, a mixed pool of MIP capture probes is added to individual genomic DNA samples in a single-well reaction. Each probe is designed with two arms flanking a targeted region, such that when a probe anneals to its targeted genomic fragment, a polymerase copies the sequence between the flanking arms, and a ligase joins this copied sequence to the probe backbone. We established a panel of 67 genes associated with CPHD in humans and mice, including new candidate genes found in the Prop1 mutants. This panel targets 693 coding exons. We analyzed 40 pediatric patients from Argentina with CPHD and relatives. In our MIP experiments, we obtained deep coverage over targeted regions, with approximately 2.1 million reads per individual, 97.6% of targeted bases reach ≥8X read depth coverage and 95.1% of bases reach ≥40X, such that up to 160 individuals could be readily pooled for sequencing on a single HiSeq lane. We believe that identifying these potential variants will make it feasible to predict clinical outcomes from genetic data, which is necessary for patient diagnosis and prognosis, and for assessing the risk of future affected individuals.