Characterization of a Novel POU1F1 Mutation Identified on Screening 160 Growth Hormone Deficiency Patients

Abstract

The objective of the study is the functional characterization of a novel POU1F1 c.605delC mutation in combined pituitary hormone deficiency (CPHD) and to report the clinical and genetic details of 160 growth hormone deficiency patients. Screening of GH1, GHRHR, POU1F1, PROP1, and HESX1 genes by Sanger sequencing was carried out in 160 trios and 100 controls followed by characterization of the POU1F1 c.605delC mutation by expression studies including site directed mutagenesis, co-transfection, protein degradation, and luciferase assays to compare the wild type and mutant POU1F1. In vitro studies showed that the POU1F1 c.605delC mutation codes for a truncated protein with reduced transactivation capacity on its downstream effectors, viz., growth hormone (GH) and prolactin (PRL) causing severe CPHD. Experiments using different protease inhibitors reveal rescue of the protein upon blockage of the lysosomal pathway that might be useful in novel drug designing using targeted approach thereby maintaining the milieu and preventing/delaying the disease. The study provides an insight into the disease causing mechanism of POU1F1 c.605delC mutation identified in a CPHD child with severe short stature and failure to thrive. It also shows mutation effect on the expression, function and turnover of protein and highlights mechanistic details by which these potent regulators may operate.

Key words

CPHD - POU1F1 - mutation screening - mutation characterization

^* Corresponding authors

• References

• 1 Zargar AH, Laway BA, Masoodi SR. et al. An aetiological profile of short stature in the Indian subcontinent. J Paediatr Child Health 1998; 34: 571-576
• 2 Dattani M, Preece M. Growth hormone deficiency and related disorders: Insights into causation, diagnosis, and treatment. Lancet 2004; 363: 1977-1987
• 3 Mullis PE. Genetics of isolated growth hormone deficiency. J Clin Res Pediatr Endocrinol 2010; 2: 52-62
• 4 Kelberman D, Rizzoti K, Lovell-Badge R. et al. Genetic regulation of pituitary gland development in human and mouse. Endocr Rev 2009; 30: 790-829
• 5 Desai MP, Mithbawkar SM, Upadhye PS. et al. Molecular genetic studies in isolated growth hormone deficiency (IGHD). Indian J Pediatr 2013; 80: 623-630
• 6 Desai MP, Mithbawkar SM, Upadhye PS. et al. Growth hormone (GH-1) gene deletions in children with isolated growth hormone deficiency (IGHD. Indian J Pediatr 2012; 79: 875-883
• 7 Birla S, Khadgawat R, Jyotsna VP. et al. Identification of novel GHRHR and GH1 mutations in patients with isolated growth hormone deficiency. Growth Horm IGF Res 2016; 29: 50-56
• 8 Birla S, Khadgawat R, Jyotsna VP. et al. Identification of Novel PROP1 and POU1F1 mutations in patients with combined pituitary hormone deficiency. Horm Metab Res 2016; 48: 822-827
• 9 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215
• 10 Huttlin EL, Ting L, Bruckner RJ. et al. The BioPlex Network: A systematic exploration of the human interactome. Cell 2015; 162: 425-440
• 11 Koch B, Schmidt C, Ploier B. et al. Modifications of the C terminus affect functionality and stability of yeast triacylglycerol lipase Tgl3p. J Biol Chem 2014; 289: 19306-19316
• 12 Alatzoglou KS, Turton JP, Kelberman D. et al. Expanding the spectrum of mutations in GH1 and GHRHR: Genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency. J Clin Endocrinol Metab 2009; 94: 3191-3199
• 13 Kelberman D, Turton JP, Woods KS. et al. Molecular analysis of novel PROP1 mutations associated with combined pituitary hormone deficiency (CPHD). Clin Endocrinol (Oxf) 2009; 70: 96-103
• 14 Kandemir N, Vurallı D, Taşkıran E. et al. Frequency of mutations in PROP-1 gene in Turkish children with combined pituitary hormone deficiency. Turk J Pediatr 2012; 54: 570-575
• 15 Rainbow LA, Rees SA, Shaikh MG. et al. Mutation analysis of POUF-1, PROP-1 and HESX-1 show low frequency of mutations in children with sporadic forms of combined pituitary hormone deficiency and septo-optic dysplasia. Clin Endocrinol (Oxf) 2005; 62: 163-168
• 16 De Rienzo F, Mellone S, Bellone S. et al. Frequency of genetic defects in combined pituitary hormone deficiency: A systematic review and analysis of a multicentre Italian cohort. Clin Endocrinol (Oxf) 2015; 83: 849-860
• 17 Miyata I, Vallette-Kasic S, Saveanu A. et al. Identification and functional analysis of the novel S179R POU1F1 mutation associated with combined pituitary hormone deficiency. J Clin Endocrinol Metab 2006; 91: 4981-4987
• 18 Hug N, Longman D, Cáceres JF. Mechanism and regulation of the nonsense-mediated decay pathway. Nucleic Acids Res 2016; 44: 1483-1495
• 19 Vellai T, Takács-Vellai K. Regulation of protein turnover by longevity pathways. AdvExp Med Biol 2010; 694: 69-80