ESPE Abstracts (2015) 84 FC11.2

Functional Characterisation of a POU1F1 Mutation Unexpectedly Associated with Isolated Growth Hormone Deficiency (IGHD): A Novel Aetiology of IGHD

Marie-Laure Sobriera,b, Yu-Cheng Tsaic, Christelle Péreza,b, Bruno Leheupd, Tahar Boucebae, Philippe Duquesnoya,b, Daria Sizovac, Stephen Liebhaberc, Nancy E Cookec & Serge Amselema,b

aInserm UMRS933 Hopital Trousseau, Paris, France; bUMRS933 Sorbonne Universités UPMC Univ Paris 06, Paris, France; cDepartment of Genetics, Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, USA; dService de Génétique clinique Pôle enfants CHU Nancy, Vandoeuvre, France; eInstitiut de Biologie Paris-Seine Plateforme Interactions Moléculaires UPMC Paris 06, Paris, France

Background: In humans, the GHN gene transcription is under the control of a Locus Control Region (LCR) enhancer, HSI, located 14.5 kb 5′ to the hGHN promoter. POU1F1, a pituitary-specific transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and the activation of GHN, PRL and TSH gene transcription. All POU1F1 mutations so far reported have been linked to a combined deficit in GH, PRL and TSH. The association of POU1F1 with three cognate sites in HSI has binding specificity and functions distinct from that to sites in responsive gene promoters. POU1F1 binding to HSI activates a domain of noncoding transcription that loops to the hGH promoter then triggers GHN transcription.

Objective and hypotheses: To explore the molecular basis of an IGHD phenotype segregating as a dominant trait within a large three-generation family.

Methods: Sequencing of candidate genes. Functional studies to decipher the mechanism underlying the consequences of the identified mutation: co-immunoprecipitation (CoIP), DNA-protein interaction by EMSA and kinetic studies by Surface Plasmon Resonance (SPR). Generation of a mouse ‘knock-in’ model of the Pou1f1 missense mutation.

Results: A POU1F1 heterozygous missense mutation (P76L) was identified in the nine affected family members. This amino-acid is highly conserved within the transactivating domain of POU1F1. A low-yield mutant protein production was observed, suggesting an impact on protein conformation. As shown by CoIP, the P76L mutation increases interactions of POU1F1 with different cofactors (LHX3, PITX1, ELK1). SPR experiments revealed increased binding affinity of the P76L protein to LCR sites. EMSA studies on the LCR and the GHN promoter sites with a mix of wt and mutant POU1F1 revealed a pattern different from that with wt alone, whereas those patterns are similar on the PRL promoter. The mutation was ‘knocked in’ to the endogenous mouse Pou1f1 locus. In heterozygotes, while levels of mutant mRNA were equivalent to wt, P76L protein was poorly expressed and failed to repress mouse GH as well as an hGH transgene expressions.

Conclusion: This is the first report of a POU1F1 mutation associated with IGHD. The data obtained unveil a novel mechanism underlying a dominant form of IGHD in humans.