Pituitary Stalk Interruption Syndrome:Follow-up Of Hormone Replacement Therapy And Studyof Its Pathogenesis By Applicating Next Generation Sequecing

Objective1. We aim to investigate the long-term benefits of growth hormone (GH) therapy in short stature adolescents and adults with pituitary stalk interruption syndrome(PSIS), which would be beneficial for future clinical applications.2. The underpinning mechanisms of sporadic PSIS patients who are in a vast majority remain elusive, necessitating a comprehensive study using systemic approaches. We aim to explore genetic mutations in sporadic patients with typical pituitary stalk interruption syndrome (PSIS), seek possible pathogenic genes and loci,provide basis for clinical diagnosis, gene therapy and genetic counselling.3. We postulate that other genetic mechanisms may be responsible for the sporadic PSIS. To test this hypothesis, we conducted a study in a family who has a PSIS patient with one unaffected sibling and healthy parents of Han Chinese using whole genome sequencing and Sanger’s sequencing.4. Cell model was constructed to validate inmapct of cell morphology, cell viability and the effect of pituitary development related protein expression level by gene silence expression of NBPF9, MUC12, TYW1B.Methods1. In this study, initial height, final height, total height gain, and GH treatment history were retrospectively investigated in 75 Chinese PSIS patients with short stature. We compared height gain between the GH treated cohort and untreated cohort and explored the impact of different GH therapy duration on height gain.2. We conducted a study in 24 PSIS patients of Han Chinese with no family history using whole exome sequencing (WES) and bioinformatic analysis was carried out to find out relevant pathways within which regulated genes were significantly enriched.3. Whole genome sequencing was performed on the four family members,bioinformatic analysis was carried out to find out relevant pathways within which regulated genes were significantly enriched, the most frequently mutations were further validated using Sanger sequencing.4. SiRNA of NBPF9, MUC12, TYW1B gene wene constructed, respectively transfected into human embryonic stem cells (H1 cell), identifying the transfection efficiency and inhibiting efficiency, observing cell morphology before and after transfection, measuring cell viability by CCK8 method, detecting pituitary associated LHX3, PITX1 gene expression in protein level using western blotting.Results1. For GH treated patients, their final height (SDS) increased from-1.99±1.91(-6.93～2.80) at bone age (BA) of 11.2 (5.0～17.0) years to -1.47 ± 1.64 (-7.82～1.05)at BA of 16.6 (8.0～18.0) years (P = 0.016). And GH treated patients had more height gain than the untreated patients0.55 (-4.55～6.46)vs.0.34(-4.53～5.90)(P = 0.011).There was a significant difference between the different GH therapy duration groups(P = 0.001): GH0 versus GH 3, P = 0.000; GH 1 versus GH 3, P = 0.028; GH 2 versus GH 3, P = 0.044.2. Using WES, a total of about 5300 mutations were detected including about 4500 point mutations and 800 InDels. We identified a group of heterozygous mutations in 92% (22/24) of the patients, and these genes are associated with Notch,Shh, Wnt signaling pathways.3. Using whole-genome sequencing, gene mutation spectrum of all four family members including structural variants, copy number variants and loss of heterozygosity.4. After silence the expression of NBPF9, MUC12, cell vitality declined, at the protein level, LHX3 expression decreased after inhibition of NBPF9, while PITX1 showed no obvious change.Conclusions1. Adult Chinese PSIS patients with short stature benefited the most from at least 12 months of GH therapy. Although patient diagnosis age was lagged behind in the developing countries, GH treatment was still effective for them and resulted in a higher final height and more height gain.2. We suggest synergy of compound mutations underpins the pathogenesis of sporadic PSIS.3. NBPF9, MUC12, TYW1B were selected for further functional verification.4.Inhibition of NBPF9 decreased pituitary development related gene LHX3 protein expression in human embryonic stem cells, the specific mechanism remains to be further discussed.