PROP-1 gene mutations in a 63-year-old woman presenting with osteoporosis and hyperlipidaemia

Introduction

Combined pituitary hormone deficiency (CPHD) is diagnosed when the production of two or more pituitary hormones is insufficient or absent. It can result either from birth trauma or asphyxia or from defects of genes controlling pituitary cell differentiation. It is mainly sporadic;¹ however, familial forms have also been described with autosomal recessive, autosomal dominant or X-linked recessive modes of inheritance.^2-4 Several developmental genes (PIT-1, PROP-1, LHX-3, LHX-4, HESX-1, SIX-6, OTX-2, PTX-2, GLI-2 and SOX-3) have been identified as important for organ commitment and cell differentiation and proliferation and have been implicated in hypopituitarism in mice and humans.^5-8

Prophet of Pit-1 (PROP-1) gene mutations as a cause of CPHD in humans was first recognised in 1998.⁹ The hormonal deficiencies in PROP-1 gene defects include growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), luteneizing hormone (LH) and follicle-stimulating hormone (FSH). In a subset of these patients, adrenocorticotropin hormone (ΑCTH) insufficiency develops as a relatively late manifestation, which thus far remains unexplained since the corticotroph cell is outside the lineage of cells affected by the PROP-1 gene.¹⁰ In addition, low values of dehydroepiandrosterone sulphate (DHEA-S) have been reported in the presence of normal pituitary-adrenal axis.¹¹

To date, at least 24 distinct mutations of the PROP-1 gene have been described, either homozygous recessive or compound heterozygous.^9,12-14 These mutations represent the most frequently reported aetiology of genetically determined CPHD.^13,15-17

As previously mentioned, patients with PROP-1 gene mutations can present with late-onset central hypocortisolism, possibly because important paracrine factors normally produced by the cells surrounding the corticotropes are absent in the pituitary of these patients, leading to progressive corticotrope cell apoptosis.^15,18,19 The physical and hormonal phenotypes of affected individuals vary widely, both within and between pedigrees, and this is often the reason for a delayed diagnosis. Pituitary morphology in patients with PROP-1 gene mutations is variable.Normal, small or enlarged pituitary gland have all been reported. Although a small pituitary gland is frequently observed, especially in oldersubjects, a significant number of young patients with PROP-1 gene mutations demonstrate pituitary enlargement.^19-21

In this paper, we report a 63-year-old female who presented with osteoporosis and hyperlipidaemia and was very short (125cm). She was found to have central hypothyroidism, low GH, low cortisol, prolactin and gonadotropins which were caused by a homozygous mutation of the PROP-1 gene. This case emphasizes the need for early aetiologic classification of short stature and long-term follow-up of patients with PROP-1 mutations in order to detect and treat promptly impaired growth and pubertal development and, importantly, the late onset of corticotropin deficiency.

Case Report

Case history

A 63-year-old female presented to the orthopaedic clinic for management of osteoporosis. Because of her extreme short stature and hyperlipidaemia refractory to three different pharmacologic agents, she was referred to the endocrine clinic for further investigation and treatment.

Physical examination showed that her height was 125 cm, the body weight 39 kg, the BMI 25 kg/m², the Breast development was Tanner Stage II and the Pubic Hair Tanner Stage II. The blood pressure was 140/60 mmHg without orthostatic hypotension. A history of absent puberty and primary amenorrhoea was reported; however, there was no history of disturbances in mental development. She mentioned having received estrogen treatment for a short period of time during her school years. Subsequently, treatment was discontinued on her own initiative and she was lost to follow-up. Her current medical status also included hypertension, hyperlipidaemia and osteoporosis, for which she was on relevant treatment with an angiotensin-converting enzyme inhibitor, pravastatin, ezetimibe, omega-3 fatty acids, risedronate and alphacalcidol. Her bone density, measured by DEXA, was diagnostic of osteoporosis in the lumbar spine [T-score (total): -3.4, Z-score (total): -1.44] and diagnostic of osteopenia in the hip [T-score (Total hip: -1.1, Neck: -1.8), Z-score (Total hip: 0.2, Neck: -0.2)] . She did not have any previous history of fractures. She was not aware of any family history of pituitary or thyroid disease; however, she mentioned that two of her female cousins (daughters of her mother’s sister) also have short stature but have never been investigated further. As far as her parents were concerned, they were born in the same village, but there was no known consanguinity between them. Her three brothers are of normal height and have no hormonal deficiencies.

Methods

Informed consent from the patient and the approval of the Hospital Ethics Committee were obtained before initiating this study.

Hormonal evaluation

A blood sample for basal hormone levels was collected at 8.00 am following an overnight fasting.

Serum TSH, Free Thyroxine (FT4), Triiodothyronine (T3), PRL, LH, and FSH were determined using the automated chemiluminescence system (ACS 180, Bayer Diagnostics Europe Ltd., Dublin, Ireland).

A glucagon test was carried out following the intramuscular administration of glucagon (1 mg). Blood samples were obtained prior to and every 30 minutes for 3 hours for measurement of cortisol and GH. Adrenal function was also investigated by measuring cortisol levels prior to and 30 and 60 min following intravenous administration of tetracosactrin (synthetic ACTH, 250μg). GH and cortisol were determined by immunoradiometric assay (kit from CIS Bio-International, Gif-sur-Yvette, France) and the chemiluminescence immunoassay (Nichols Advantage, Nichols Institute Diagnostics, San Juan Capistrano, CA, USA).

Genomic analysis of the PROP-1 gene

DNA was extracted from peripheral blood leucocytes employing the Maxwell 16 Instrument AS2000, automated purification system (Promega Corporation, Madison, USA). Exons 2 and 3 and their flanking intronic sequences of the PROP-1 gene were PCR amplified and directly sequenced in the forward and reverse direction on an automated sequencer (ABI 3100 Avant, Applied Biosystems). PCR conditions and primers used for amplification and sequencing have been previously described.¹⁹

Results and clinical course

Laboratory data are shown in Tables 1 and 2. Serum levels of FT4 and T3 were very low with inappropriately normal levels of TSH, indicating central hypothyroidism and a possible additional cause for our patient’s hyperlipidaemia. Creatinine kinase (CK) value was high, and 25-hydroxycholecalciferol levels (25OHD₃) were low.

FSH and LH values were low despite the patient’s menopausal age. PRL levels were low and plasma ACTH level was at the lower limit of the normal range [8.9 pg/ml (NV: 7-52pg/ml)]. Peak growth hormone and cortisol values during glucagon test were <0.05ng/ml (NV ≥10ng/ml) and 11.6μg/dl (NV ≥18μg/dl), respectively. Plasma cortisol at 08.00 was low and 60 min post Synacthen was 16.2 μg/dl (NV ≥18 μg/dl). These results showed inadequate response of cortisol secretion to the administration of either glucagon or synthetic ACTH. Although the insulin tolerance test (ITT) is the most powerful test to evaluate the hypothalamic-pituitary-adrenal (HPA) axis, it was not performed as the patient did not give her consent.

Sequencing of exons 2 and 3 of the PROP-1 gene revealed that the patient was homozygote for the previously described exon 2 mutation, p.R73H,¹⁵ thus clarifying the aetiology of the patient’s CPHD. Replacement therapy with hydrocortisone (10 mg am, 5 mg pm) was initiated followed by replacement therapy with L-thyroxine (50 μg/day) and 25OHD₃ (1200 units/day). Her hypolipidaemic treatment was discontinued and lipid levels decreased (Total cholesterol: 173 mg/dl, high density lipoprotein (HDL): 43, triglycerides (TGL): 142 mg/dl). Under thyroxine treatment, circulating concentrations of FT4 and T3 stayed within the normal range [FT4 1.04 ng/dl (NV: 0.7-1.85 ng/dl) and T3 1.01 ng/ml (NV: 0.5-1.4 ng/ml)]. She has been doing well and feeling more energetic as a sense of general fatigue, which had not been mentioned, has improved. Unfortunately, MRI of the pituitary gland was not possible since the patient refused because of claustrophobia.

Discussion

CPHD is a rare disorder characterized by impaired production of GH and one or more of the other anterior pituitary hormones. Two pituitary transcription factors have been mainly implicated in the pathogenesis of this condition: the human homologue of the mouse Pit-1 (POU1F1)^5,23 and PROP-1.^6,24 The PROP-1 gene is necessary for POU1F1 gene expression and consequently for the differentiation of the POU1F1-dependent cell lineages (somatotropes, lactotropes and caudomedial thyrotropes) as well as for the differentiation of the gonadotropes.^6,24 Consequently, mutations of the PROP-1 gene are responsible for the absence of the Pit1-dependent cell lineages and in addition for reduced numbers of gonadotropes, resulting in the deficiency of GH, PRL, TSH, FSH and LH.^{9,13,20,25-27}

The human PROP-1 gene encodes a protein of 226 aminoacids and its mutations account for the majority of cases of recessively inherited CPHD in persons of European origin.^9,12,13 The first examples of PROP-1 mutations in humans with CPHD were reported in 1998,⁹ following the report of Sornson et al in 1996 of the Ames dwarf mice mutation.⁶ Thus far, at least 24 distinct mutations of the PROP-1 gene have been identified in over 170 patients. The most common types of mutation are a two-base-pair deletion in codon 101 (301-302delAG)²⁸ and a one-base-pair deletion in codon 50 (150delA). Several other missense, nonsense and splice site mutations have been described, such as p.R73C, p.R73H, p.F88S, p.F117I, p.R120C, p.R120H, p.R99X, p.Q83X.^{9,13,15,21,22,26,29} Patients with PROP-1 gene mutations exhibit some phenotypic variability, involving age of onset of hormone deficiencies,^30,31 pituitary size^9,28,31,32 and cortisol secretion.^9,28

In a number of patients with PROP-1 gene mutations, late-onset hypocortisolism has been detected,^15,18 with a tendency for ACTH and cortisol deficiencies to emerge with advancing age.^20,27,33 Such a progressive ACTH deficiency with age is not observed in Ames dwarf mice.⁶ Moreover, it has not been linked to a specific mutation. This suggests that a more complex mechanism than simple failure of embryonic development of the corticotroph cell lineage may be involved, as proposed by Pernasetti et al.²⁷

In our patient with CPHD a p.R73H homozygous mutation in the 2^nd exon of the PROP-1 was identified. The patient remained undiagnosed and untreated till the age of 63 years, when she presented with osteoporosis and hyperlipidaemia refractory to medical treatment. Her bone density was diagnostic of osteroporosis in the lumbar spine and osteopenia in the hip, both due to hypogonadotropic hypogonadism and 25OHD₃ deficiency. Her serum levels of FT4 and T3 were very low with inappropriately normal levels of TSH, indicating the diagnosis of central hypothyroidism and the cause of her hyperlipidaemia that was refractory to medical treatment. The levels of other pituitary hormones, such as PRL, FSH and LH, and the levels of DHEA-S were also low, confirming the diagnosis of anterior lobe pituitary insufficiency.⁹ Her short stature, lack of pubertal development as well as primary amenorrhoea indicated that her pituitary insufficiency was manifested early in life. Dynamic pituitary testing with glucagon test revealed GH as well as ACTH deficiency. Although ITT remains the gold standard test for assessing the entire HPA axis, several authors advocate the Short Synacthen Test (SST) with appropriate cut-offs as the first line test.^34-36 While controversy exists about the cut-offs and diagnostic reliability, the potential for missing subtle or recent HPA defects remains a possibility.³⁷ Our patient had a longstanding hypopituitarism and additionally she declined to have an ITT, therefore we decided to perform a SST. Despite the SST result being consistent with partial secondary hypoadrenalism due to long-term ACTH insufficiency, this patient had never had any clinical or laboratory findings of hypoadrenalism, such as orthostatic hypotension, hyponatraemia or hypoglycaemia. This may be due to the well known fact that patients with incomplete hypocortisolism may experience severe symptoms only during periods of stress and remain undiagnosed for long periods. In view of the above, we decided to start cortisol replacement prior to thyroxine treatment in order to prevent the life-threatening features of adrenal insufficiency that may become apparent in the event of severe physical and psychological stress. Pituitary size of the patient is not known as she refused to have a pituitary MRI due to claustrophobia.

The mutation p.R73H of the PROP-1 gene was first described in 2001 by Vallette-Kasic et al.¹⁵ Five years later, there were two more patients found to have the same mutation without any family history of CPHD.⁸ Our patient is apparently the oldest to be reported so far with CPHD due to the above mutation. Although she does not have any known family history of CPHD, she admitted having two cousins of similar age with short stature who have not been investigated.

In conclusion, we present a female patient with short stature, hyperlipidaemia refractory to medical treatment and osteoporosis who was diagnosed with hypopituitarism due to a PROP-1 gene mutation at the age of 63. In addition to GH, PRL, TSH and gonadotropin deficiency, our patient also had partial cortisol insufficiency. Our case illustrates the importance of investigating patients with short stature to identify the hormonal and genetic cause and offer the most appropriate treatment. As the clinical phenotypes of human PROP-1 mutations may be variable and modified by time, it is important to assure etiologic classification and long-term follow-up of such patients in order to improve quality of life as well as morbidity and mortality.

Conflicts of interest

The authors declare that there is no conflict of interest.

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