MON-712 Restoration of Growth and Fertility in Zebrafish (Danio Rerio) Model with PROP1 Knockout Generated by CRISPR/Cas9 Genomic Editing

Abstract Introduction: Hypopituitarism is defined as the deficiency of one or more pituitary hormones and can occur due to pathogenic allelic variants in transcription factors involved in pituitary development. PROP1 gene is responsible for progenitor cell migration from the marginal zone to the anterior lobe, and its terminal differentiation into corticotropes and gonadotropes cell lines besides somatotropes, lactotropes and thyrotropes due to POU1F1 (also known as PIT1) activation. In humans, mutations in the PROP1 gene are the most common cause of congenital hypopituitarism with GH, TSH, LH/FSH, and progressive ACTH deficiencies. A dwarf phenotype with short stature, pituitary hormone deficiency, and infertility has been described in humans and Ames mice lineage harboring mutations in the PROP1/Prop1 gene. Another valuable animal model used in basic research is the zebrafish (Danio rerio) due to a high homology in neuroendocrine functioning. To test the potential of this model, in our previous study, a 32bp insertion carrying a stop codon was directed into the second exon of prop1 with CRISPR/Cas9, establishing a homozygous mutant strain (prop1mut). Objective: To characterize the phenotype and expression patterns of transcription factors and hormones in the zebrafish prop1mut lineage. Methods: prop1, pit1, and gh1 mRNA levels were analyzed during embryonic development at 24 and 72 hours post-fertilization (hpf). RNA from 30 pooled embryos was extracted using DirectZol RNA Miniprep. cDNA was synthesized from 1ug of total RNA using High-Capacity cDNA Reverse Transcription Kit and qPCR was performed using SYBR Green PCR Master Mix. Gene expression was normalized to ef1a and the prop1mut group was compared with the control wild type group (WT). Animals were kept in the tanks at a density of 15 animals/liter and images were acquired at 13 and 20 days post fertilization (dpf) after brief anesthetization using a stereomicroscope and measured in ImageJ software to determine the larval standard length from nose to the end of the spinal cord. Results: At 24 and 72hpf, prop1mut embryos expressed the altered prop1 mRNA at similar levels to the prop1 expression observed in WT. Lower pit1 expression in prop1mut embryos was observed at both periods (p<0.01). Albeit in low levels, similar gh1 expression was observed in both lineages at 24hpf, and prop1mut embryos presented lower gh1 expression at 72hpf (p<0.001). prop1mut larvae presented a significant decrease in size at 13dpf (p<0.001) but not at 20dpf. Conclusion: In this study, the prop1mut zebrafish model exhibited a dwarf phenotype during larval development associated with diminished pit1 and gh1 expression during the embryonic stage. Additionally, in the juvenile stage, the development rate in prop1mut animals was restored, presenting similar standard lengths observed in WT animals.

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Late onset adrenocorticotrope deficiency in combined pituitary hormone deficiency caused by a mutation of the PROP1 gene

Endocrine Abstracts ◽

10.1530/endoabs.56.p721 ◽

2018 ◽

Author(s):

Silvia Paredes ◽

Olinda Marques ◽

Marta Alves

Keyword(s):

Late Onset ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Pituitary Hormone Deficiency ◽

Combined Pituitary Hormone Deficiency ◽

Prop1 Gene

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MECHANISMS IN ENDOCRINOLOGY: An update in the genetic aetiologies of combined pituitary hormone deficiency

Acta Endocrinologica ◽

10.1530/eje-15-1095 ◽

2016 ◽

Vol 174 (6) ◽

pp. R239-R247 ◽

Cited By ~ 26

Author(s):

Frederic Castinetti ◽

Rachel Reynaud ◽

Alexandru Saveanu ◽

Nicolas Jullien ◽

Marie Helene Quentien ◽

...

Keyword(s):

Transcription Factors ◽

G Protein Coupled Receptors ◽

Hormone Deficiency ◽

Immunoglobulin Superfamily ◽

Pituitary Hormone ◽

Pituitary Hormone Deficiency ◽

Combined Pituitary Hormone Deficiency ◽

Signalling Molecules ◽

Genes Encoding ◽

G Protein Coupled

Over the last 5 years, new actors involved in the pathogenesis of combined pituitary hormone deficiency in humans have been reported: they included a member of the immunoglobulin superfamily glycoprotein and ciliary G protein-coupled receptors, as well as new transcription factors and signalling molecules. New modes of inheritance for alterations of genes encoding transcription factors have also been described. Finally, actors known to be involved in a very specific phenotype (hypogonadotroph hypogonadism for instance) have been identified in a wider range of phenotypes. These data thus suggest that new mechanisms could explain the low rate of aetiological identification in this heterogeneous group of diseases. Taking into account the fact that several reviews have been published in recent years on classical aetiologies of CPHD such as mutations ofPOU1F1orPROP1, we focused the present overview on the data published in the last 5 years, to provide the reader with an updated review on this rapidly evolving field of knowledge.

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Selenium status affects selenoprotein expression, reproduction, and F1 generation locomotor activity in zebrafish (Danio rerio)

British Journal Of Nutrition ◽

10.1017/s000711451300439x ◽

2014 ◽

Vol 111 (11) ◽

pp. 1918-1931 ◽

Cited By ~ 17

Author(s):

Sam Penglase ◽

Kristin Hamre ◽

Josef D. Rasinger ◽

Staale Ellingsen

Keyword(s):

Locomotor Activity ◽

Reproductive Success ◽

Mrna Expression ◽

Danio Rerio ◽

Expression Patterns ◽

Maximum Growth ◽

Cellular Growth ◽

Mrna Levels ◽

Gpx Activity ◽

Se Status

Se is an essential trace element, and is incorporated into selenoproteins which play important roles in human health. Mammalian selenoprotein-coding genes are often present as paralogues in teleost fish, and it is unclear whether the expression patterns or functions of these fish paralogues reflect their mammalian orthologues. Using the model species zebrafish (Danio rerio; ZF), we aimed to assess how dietary Se affects key parameters in Se metabolism and utilisation including glutathione peroxidase (GPX) activity, the mRNA expression of key Se-dependent proteins (gpx1a, gpx1b, sepp1a and sepp1b), oxidative status, reproductive success and F1 generation locomotor activity. From 27 d until 254 d post-fertilisation, ZF were fed diets with graded levels of Se ranging from deficient ( < 0·10 mg/kg) to toxic (30 mg/kg). The mRNA expression of gpx1a and gpx1b and GPX activity responded in a similar manner to changes in Se status. GPX activity and mRNA levels were lowest when dietary Se levels (0·3 mg/kg) resulted in the maximum growth of ZF, and a proposed bimodal mechanism in response to Se status below and above this dietary Se level was identified. The expression of the sepp1 paralogues differed, with only sepp1a responding to Se status. High dietary Se supplementation (30 mg/kg) decreased reproductive success, while the offspring of ZF fed above 0·3 mg Se/kg diet had lower locomotor activity than the other groups. Overall, the novel finding of low selenoprotein expression and activity coinciding with maximum body growth suggests that even small Se-induced variations in redox status may influence cellular growth rates.

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A novel PROP1 gene mutation (157delA) in Japanese siblings with combined anterior pituitary hormone deficiency

Clinical Endocrinology ◽

10.1111/j.1365-2265.2004.02147.x ◽

2004 ◽

Vol 61 (5) ◽

pp. 635-640 ◽

Cited By ~ 16

Author(s):

Ke-ita Tatsumi ◽

Kiyoshi Kikuchi ◽

Kumi Tsumura ◽

Nobuyuki Amino

Keyword(s):

Gene Mutation ◽

Anterior Pituitary ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Pituitary Hormone Deficiency ◽

Anterior Pituitary Hormone ◽

Prop1 Gene

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The PROP1 2-Base Pair Deletion Is a Common Cause of Combined Pituitary Hormone Deficiency1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.9.5142 ◽

1998 ◽

Vol 83 (9) ◽

pp. 3346-3349 ◽

Cited By ~ 2

Author(s):

Joy D. Cogan ◽

Wei Wu ◽

John A. Phillips ◽

Ivo J. P. Arnhold ◽

Ana Agapito ◽

...

Keyword(s):

Human Subjects ◽

Polymorphic Marker ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Degree Relative ◽

Exon 2 ◽

Base Pair Deletion ◽

Stimulation Tests ◽

Sporadic Cases ◽

Prop1 Gene

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301–302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301–302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301–302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301–302delAG deletion suggests that rather than being inherited from a common founder, the 301–302delAG may be a recurring mutation.

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The c.301_302delAG PROP1 gene mutation in Romanian patients with multiple pituitary hormone deficiency

Journal of Pediatric Endocrinology and Metabolism ◽

10.1515/jpem-2014-0289 ◽

2015 ◽

Vol 28 (9-10) ◽

Cited By ~ 1

Author(s):

Cecilia Lazea ◽

Paula Grigorescu-Sido ◽

Radu Popp ◽

Marie Legendre ◽

Serge Amselem ◽

...

Keyword(s):

High Frequency ◽

Bone Age ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Homozygous State ◽

Pituitary Hormone Deficiency ◽

Homozygous Genotype ◽

Familial Form ◽

History Of ◽

Prop1 Gene

AbstractTo establish the frequency of the c.301_302 delAG mutation of theSomatic assessment, hormonal test, bone age, magnetic resonance imaging of the pituitary gland, and molecular diagnosis were performed in 26 patients with MPHD (7 patients with familial form of MPHD and 19 patients with sporadic form of MPHD).The c.301_302delAG mutation was detected in the homozygous state in 10 patients belonging to 5 unrelated families (7 patients with familial history of MPHD and 3 patients with sporadic form of MPHD). Those 10 patients presented variable pituitary hormone deficiency and pituitary morphology.The c.301_302delAG homozygous genotype had a high frequency of 38% (10/26), reaching 100% (7/7) in group with familial cases of MPHD and 16% (3/19) in group with sporadic forms of MPHD.

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Molecular analysis of the PROP1 and HESX1 genes in patients with septo-optic dysplasia and/or pituitary hormone deficiency

Arquivos Brasileiros de Endocrinologia & Metabologia ◽

10.1590/s0004-27302010000500009 ◽

2010 ◽

Vol 54 (5) ◽

pp. 482-487 ◽

Cited By ~ 3

Author(s):

Juliana B. Cruz ◽

Vania S. Nunes ◽

Sueli A. Clara ◽

Denise Perone ◽

Peter Kopp ◽

...

Keyword(s):

Gene Mutations ◽

Genetic Alterations ◽

Hormone Deficiency ◽

Pituitary Hormone ◽

Pituitary Hormone Deficiency ◽

Exon 1 ◽

Genetic Mechanisms ◽

Direct Sequence Analysis ◽

Prop1 Gene ◽

Pathogenic Process

OBJECTIVE: The present study aimed at evaluating the PROP1 and HESX1 genes in a group of patients with septo-optic dysplasia (SOD) and pituitary hormone deficiency (combined - CPHD; isolated GH deficiency - GHD). Eleven patients with a clinical and biochemical presentation consistent with CPHD, GHD or SOD were evaluated. SUBJECTS AND METHODS: In all patients, the HESX1 gene was analyzed by direct sequence analysis and in cases of CPHD the PROP1 gene was also sequenced. RESULTS: A polymorphism (1772 A > G; N125S) was identified in a patient with SOD. We found three patients carrying the allelic variants 27 T > C; A9A and 59 A > G; N20S in exon 1 of the PROP1 gene. Mutations in the PROP1 and HESX1 genes were not identified in these patients with sporadic GHD, CPHD and SOD. CONCLUSION: Genetic alterations in one or several other genes, or non-genetic mechanisms, must be implicated in the pathogenic process.

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Functional SNPs within the Intron 1 of the PROP1 Gene Contribute to Combined Growth Hormone Deficiency (CPHD)

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2012-1527 ◽

2012 ◽

Vol 97 (9) ◽

pp. E1791-E1797 ◽

Cited By ~ 3

Author(s):

Michela Godi ◽

Simona Mellone ◽

Luigi Tiradani ◽

Rita Marabese ◽

Claudio Bardelli ◽

...

Keyword(s):

Regulatory Element ◽

Hormone Deficiency ◽

Luciferase Reporter ◽

Pituitary Hormone ◽

Mcf7 Cells ◽

Luciferase Reporter Gene ◽

Risk Alleles ◽

Intron 1 ◽

Sporadic Cases ◽

Prop1 Gene

Context: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. Objective: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. Methods and Patients: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. Results: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10−4) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10−4) and conferred a carrier risk of 4.19 (P = 1.2 × 10−4). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10−6; P = 5.5 × 10−4, respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. Conclusions: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.

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