Introduction

Cushing's disease is a very common endocrinopathy in dogs, generally caused by an ACTH-secreting corticotroph adenoma (Feldman & Nelson 2004). Although the disease has been described in dogs for 60 years, little is known about its underline pathogenesis (vanWijk et al. 1997). There is a high incidence of Cushing syndrome in poodles and familial pituitary hypercortisolism have been identified suggesting a genetic involvement. In the pituitary gland, Tpit is an obligate transcription factor for expression of the pro-opiomelanocortin (POMC) gene and for terminal differentiation of the corticotroph lineage (Pulichino et al. 2003). Tpit, also described as Tbx19, belongs to the Tbox family of transcription factors and is present only in pituitary POMC expressing lineages, the corticotrophs and melanotrophs (Lamolet et al. 2001). Loss-of-function mutations of Tpit are associated with neonatal isolated corticotropin deficiency (Drouin et al. 2007). We hypothesized that Tpit gain of function mutations would be involved in the pathogenesis of corticotroph tumors in poodle dogs.

Objectives

The aim of the present study was to screen the Tpit gene for mutations in genomic DNA from poodles with Cushing's disease.

Materials and Methods

Forty two poodle dogs (34 female), with median age of 9.29 +/-2.98 years, with Cushing's disease were studied. The diagnosis was based on clinical signs (polyphagia, polyuria, polydipsia, abdomen enlargement, panting, pyoderma), cortisol levels > 1.4 μg/dl after dexamethasone suppression test, plasmatic ACTH levels > 17 pg/ml, bilateral adrenal enlargement at abdominal ultrasound, hypertriglyceridemia, hypercholesterolemia, hyperphosphatasemia, hyposthenuria. Genomic DNA was isolated from peripheral blood, amplified by PCR, using inotropic primers to amplify 8 exons, and submitted to automatic sequence. The coding sequence was compared to the canine Boxer genomic DNA sequence available at NCBI site.

Results

We found the heterozygous allelic variation p.Ser343Gly (AGC > GGC) in exon 7 in 2 unrelated dogs (n = 2/42). To investigate if this variation is a mutation or a single nucleotide polymorphism, we studied fifty normal female dogs with mean age 9.38 +/-2.76 years, similarly to Cushing's dogs group. An enzymatic digestion with MSP I restriction enzyme was performed in a PCR fragment from exon 7. The allelic variation p.Ser343Gly was found in 2 alleles in the control group.

Discussion and Conclusions

Tpit, also described as Tbx19, belongs to the Tbox family of transcription factors. Genes encoding T-box factors are present in all vertebrates and several diseases have been associated with mutations of such genes in humans (Vallette-Kasic et al. 2003). In gain-of-function experiments, Tpit induces POMC expression in undifferentiated pituitary cells, indicating that it can initiate differentiation into POMC expressing lineages. Also, Tpit mutations were found in patients with isolated deficiency of pituitary POMC-derived ACTH, in support of an essential role of Tpit for differentiation of the pituitary POMC lineage (Lamolet et al. 2001). Tpit is the only currently known transcription factor selectively expressed in human normal and adenomatous corticotrophs (Vallette-Kasic  2003). Because of this, contribution of this protein to the pathogenesis of corticotropinomas has been studied. In the present study, the allelic variation leading to p.Ser343Gly (AGC > GGC) in exon 7 in heterozygous state was considered a single nucleotide polymorphism, as it was found in 4.7% (n = 2/42) of our cohort and also in 4% (n = 2/50) of the control group. According to it, a previous report did not find mutations in Tpit screening adenomatous tissue of 14 dogs with corticotroph adenomas (Hanson et al. 2008) as well as in 8 human corticotroph macroadenomas (Bucciarelli et al. 2005). We concluded that a gain of function mutation of Tpit gene is not likely to play a major role in the general pathogenesis of canine corticotroph adenomas in our cohort.

References

1.  Feldman EC, Nelson RW. 2004. Canine and feline endocrinology and reproduction. 3rd ed. Philadelphia: W.B. Saunders, 1089 p.

2.  vanWijk PA, Rijnberk A, Croughs RJ, Meij BP, van Leeuwen IS, Sprang EP et al. 1997. Molecular screening for somatic mutations in corticotrophic adenomas of dogs with pituitary-dependent hyperadrenocorticism. Journal of Endocrinology Investigation 20:1-7.

3.  Pulichino AM, Vallette-Kasic S, Tsai JP, Couture C, Gauthier Y, Drouin J. Tpit determines alternate fates during pituitary cell differentiation. 2003. Gene Development, 17:738-47.

4.  Lamolet B, Pulichino AM, Lamonerie T, Gauthier Yves, Thierry B, Enjalbert A, Drouin J. 2001. A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoprotein. Cell, 104: 849-59.

5.  Vallette-Kasic S, Figarella-Branger D, Grino M, Pulichino AM, Fufour H, Grisoli F, Enjalbert A, Drouin J, Brue T. 2003. Differential Regulation of proopiomelanocortin and pituitary restricted transcription factor (Tpit), a new marker of normal and adenomatous human corticotrophs. The Journal of Clinical Endocrinology & Metabolism, 88 (7): 3050-56.

6.  Hanson JM, Mol JA, Leegwater PAG., Bilodeau S, Drouin J, Meij BP. 2008. Expression and mutation analysis of Tpit in the canine pituitary gland and corticotroph adenomas. Domestic animal endocrinology, 34:217-22.

7.  Bucciarelli LG, Giraldi FP, Cavagnini, F. 2005. No mutations in Tpit, a corticotroph-specific gene, in human tumoral pituitary ACTH-secreting cells. Journal of Endocrinology Investigation 28: 1015-18.