Long-Term Follow-Up of Combined Pituitary Hormone Deficiency in Two Siblings With a Prophet of Pit-1 Gene Mutation

By Georgopoulos, Neoklis A; Katsikis, Ilias; Giamalis, Petros; Koika, Vasiliki; Et al

Abstract

Combined pituitary hormone deficiency (CPHD) is a rare disorder resulting from an impaired pituitary function due to different causes, characterized by impaired secretion of growth hormone (GH) and one or more of the other anterior pituitary hormones. To date, 16 distinct human Prophet of Pit-1 (Prop1) gene mutations have been identified in patients with CPHD, inducing a phenotype involving GH, follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin and thyroid-stimulating hormone (TSH), and rarely adrenocorticotropic hormone, deficiency. Herein we present two siblings of different sexes from a family with parental consanguinity presenting the 301-302delAG mutation in the Prop1 gene. The female presented failure of growth from the age of 6 years and was treated for 10 years with GH, ending in a final height (standard deviation score) of -0.28. TSH deficiency was manifested after the initiation of GH and was treated with thyroxine while puberty was initiated with conjugated estrogens. The male presented TSH deficiency since childhood, treated with thyroxine, and growth failure at the age of 14 years, treated for a period of 2 years with GH. Puberty was initiated with increasing doses of testosterone, while human chorionic gonadotropin was added in order to achieve increased testicular volume. In conclusion, these two siblings of different sexes with CPHD carrying the 301-302delAG mutation in the Prop1 gene presented a variable phenotype characterized by GH, TSH, LH and FSH deficiency.

Keywords: Hypogonadotropic hypogonadism, PROP-1, Prophet of Pit- 1, hypopituitarism, combined pituitary hormone deficiency

Introduction

Combined pituitary hormone deficiency (CPHD) is a rare disorder resulting from an impaired pituitary function due to different causes, characterized by impaired secretion of growth hormone (GH) and one or more of the other anterior pituitary hormones. The incidence is approximately 1/8000 newborns. Most cases occur sporadically; however, approximately 10% of the patients have an affected first-degree relative following an autosomal recessive, autosomal dominant or X-linked recessive inheritance pattern [1].

In congenital forms of CPHD, genetic alterations of several pituitary transcription factors (PIT1, PROP1, LHX3, LHX4, HESX1, PITX1, PITX2) have been recognized over the past 12 years, although most genetic causes of CPHD still remain unidentified [2].

The Prophet of Pit-1 (Prop1} gene is a paired-like homeobox gene mapped to chromosome 5q in humans. This gene consists of three exons and encodes a 223-amino-acid nuclear protein that contains a C- terminal transactivation domain and a paired-like homeodomain with three putative α helices [3,4] A naturally occurring homozygous mutation within Prop1 was first described in the Ames dwarf mouse, resulting in the substitution of a serine residue by proline (S83P) in the α1 helix of the homeodomain of the PROP1 paired-like transcription factor [5]. To date, 16 distinct human Prop1 mutations have been identified [6,7]. Pit1 and Prop1 mutations induce a similar phenotype involving deficiency of GH, prolactin (PRL) and thyroid-stimulating hormone (TSH). In addition, Propl mutations also induce FSH and LH deficiency causing hypogonadotropic hypogonadism and rarely adrenocorticotropic hormone (ACTH) deficiency with hypocortisolism [8].

In the present study we present the clinical followup from childhood to adolescence of two siblings of different sexes with CPHD caused by a mutation in the Propl gene.

Materials and methods

Assays for hormone determinations

Serum FSH, LH, PRL, estradiol, TSH, triiodothyronine (T3) and thyroxine (T4) were determined by enzyme-linked immunoassay using commercially available kits (Mercodia AB, Uppsala, Sweden). Serum testosterone, progesterone and dehydroepiandrosterone sulfate were measured with a radioimmunoassay method using commercially available kits (Radioisotopic Kit; Diagnostic Systems Laboratories, Webster, TX, USA).

Genetic testing

Genomic DNA was extracted from peripheral blood leukocytes samples of the two siblings as well as from their parents by the standard phenol/chloroform procedure. Each of the three exons of the Propl gene was amplified by polymerase chain reaction (PCR). The following pairs of primers were used: exon 1, 5′-GAG CTG CGG AAG CAG AGA AAT CTC A-3′ (sense primer) and 5′-AGA GGT AAC TGT CTC ACA TCC CCA C-3′ (antisense primer); exon 2, 5′-CAC TGA GCG CAA TCC CGG GAC- 3′ (sense primer) and 5′-GAG ATG AGG CCT GTG TCT GGT GA-3′ (antisense primer); exon 3, 5′CTC TTG TCA TTG GAG TAG GGT GTC A-3′ (sense primer) and 5′-CAG ACT TCC TCC ACT AAT CAC CCC A-3′ (antisense primer). PCR was performed in a total volume of 100 μl, using 200 ng genomic DNA as template, 3 mM MgCl2, 10 pmol each appropriate primer, 2 pmol each of the four dNTP, and 1 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). Reactions were carried out in a PTC-200 thermocycler (MJ Research Inc., Waltham, MA, USA) using the following parameters (33 cycles): DNA denaturation at 94C for 10 min in the first cycle and for 30 s in all subsequent cycles, annealing at 56C for 30 s and extension at 72C for 30 s, with a final elongation for 10 min. Amplification was confirmed by agarose gel electrophoresis. Subsequently, all PCR fragments were purified by NucleoSpin Extract (Macherey-Nagel GmbH & Co. KG, Dren, Germany) and were subjected to bidirectional DNA automated sequence analysis (Lark Technologies, Takeley, UK). Each potential mutation or deletion was confirmed by a second independent PCR amplification and sequencing.

Results

The patients were referred to the Division of Endocrinology and Human Reproduction, second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki for evaluation of delayed puberty.

Family history

The two probands were the only children in the family. The pedigree of the family showed parental consanguinity. Both grandmothers were step-sisters (from the same mother and different fathers). There was no evidence for CPHD or hypogonadism in any other member of the family except the probands. The reported target height (TH) was estimated using the mid-parental height as an index of genetic predisposition to adult height. The equation used for reported TH was: TH = (father’s height 13 + mother’s height)/2.9. Father’s height was 185 cm and mother’s height 159 cm.

Endocrinological evaluation

The first proband to be examined was the female, who was evaluated for short stature at the age of 6 years at the Department of Pediatrics of Ippokration Hospital of Thessaloniki. Physical examination revealed height 95 cm (standard deviation score (SDS) – 3.79) [9]. GH secretion was evaluated by provocative testing with clonidine. Blood was collected every 30 min for 2 h following clonidine administration and serum GH levels remained below 1 ng/ ml. Thereafter she was treated with subcutaneous GH injections for a period of 10 years and her final height reached 160 cm (SDS -0.28) [9], while her target height was 165.5 cm (SDS 0.53) [10]. Three months after the initiation of GH treatment central hypothyroidism was detected with low T3 and T4 and TSH = 0-01 mIU/ml, and she received thyroxine replacement therapy. At the age of 15 years, as she showed no signs of pubertal development (breast development: Tanner stage I) [11], she started treatment with increasing doses of conjugated estrogens. She menstruated normally on a cyclic preparation containing conjugated estrogens (0.625 mg) and medroxyprogesterone (10 mg). According to Tanner stages of breast and pubic hair development [11], at her last visit she showed stage V breast development and stage V pubic hair development. External genitalia were those of a normal female, without clitoris enlargement. She was clinically euthyroid with a non-palpable thyroid gland. Her hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotrope reserve were evaluated by the clomiphene citrate test and GnRH test, respectively, and GH and cortisol by the insulin tolerance test. Data are shown in Table I and reveal no response of LH, FSH, PRL and GH as well as a normal response of cortisol. Magnetic resonance imaging (MRI) and computed tomography (CT) of the hypothalamic-pituitary area were normal.

Her brother was first evaluated at the age of 14 years for deterioration of growth. Physical examination revealed height 143.5 cm (SDS -2.59) [9], while up until that time his linear growth had constantly kept above the 5th percentile. He started GH treatment for a period of 2 years and his final height was 175 cm (SDS + 0.05) [9], while his target height was 178.5 cm (SDS 0.59) [10]. Central hypothyroidism was detected during childhood and he received thyroxine replacement therapy. Pubertal development was achieved by intramuscular injections of increasing doses of testosterone enanthate up to the dose of 250 mg monthly. Finally, he was treated with human chorionic gonadotropin (hCG) 5000 IU/week for an additional period of 6 months to achieve a reasonable increase in testicular volume. At his last visit his pubic hair development was Tanner stage VII, and his axillary hair was fully developed. His external genitalia were those of a normal male, and testi\cular volume was 8 ml. He was clinically euthyroid with a non-palpable thyroid gland. His hypothalamic GnRH and pituitary gonadotrope reserve were evaluated by the clomiphene citrate test and GnRH test, respectively, and GH and cortisol by the insulin tolerance test. Data are shown in Table II and reveal no response of LH, FSH, PRL and GH as well as a normal response of cortisol. Challenge with hCG challenge revealed a normal testosterone response (Table II). MRI and CT of the hypothalamic-pituitary area were normal.

Table I. Hormonal evaluation of the female sibling.

Genetic testing

The Prop1 gene mutation detected in the two siblings was the 301- 302delAG (Figure 1). The precise location of the 2-bp GA or AG deletion in the sequence is ambiguous, so any combination of a GA or AG deletion could yield the same results. The phenotypical normal parents were found to be heterozygous for a wild-type allele and a mutated one.

Discussion

We have identified a common recessive mutation, a two-base deletion in exon 2 (301-302delAG), in a Greek family with two siblings with CPHD. The deletion causes a frameshift and if translated disrupts the paired-like homeodomain required for DNA- protein interaction, leading to complete loss of the mutated protein’s DNA-binding activity. As both siblings were carrying the mutation in homozygosity while their parents harbored it in heterozygosity, this was presumably the causative mutation for CPHD. The 301-302delAG is the most frequently found genetic defect in CPHD, identified in 53% of familial and 62% of sporadic cases reported in five different large series [7,8,12-15]. This mutational hot spot is particularly common among mainly Caucasian populations such as Russians, Swiss, Polish, Turks and Greeks, as well as among Latin American populations [7,8,12-17], while other mutations predominate in Far-East Asian populations [18,19]. The two first reports describing the 301-302delAG mutation and its consequences were those of Wu [20] and Nogueira [21] and co-workers.

Table II. Hormonal evaluation of the male sibling.

The clinical phenotype of patients with CPHD due to Propl mutations varies not only between different gene mutations but also even among siblings with the same mutation [8,22,23]. For instance, GH and TSH deficiencies were diagnosed at different ages (5.5-10.8 years) among patients carrying the same molecular defect [22], while in another study the age at diagnosis ranged from 9 months to 8 years [23]. The typical patient with CPHD due to a Propl gene defect presents an acquired progressive failure of GH secretion manifested by a progressive impairment in growth that leads to profound growth failure if untreated. Central hypothyroidism develops after GH deficiency in 80% of patients, while, in contrast to patients with a Pit1 mutation who usually manifest normal puberty, in most cases a failure to enter puberty spontaneously is noted [8]. Growth failure is usually noted during early childhood as in most cases growth during infancy parallels the lower limits of the normal curve. The median age at diagnosis is from 6 to 8 years of age. Most patients with the 301302delAG mutation present a normal postnatal period and normal growth during infancy, with growth failure being manifested between the ages of 3 and 7 years, mostly by the age of 7 years. Indeed, our female patient was diagnosed with GH deficiency at 6 years of age, although her brother, who notably is carrying the same mutation, continued to grow close to normal until 14 years of age, when GH treatment had to be initiated. On the contrary, TSH deficiency was manifested early in childhood for both probands.

Figure 1. (A) Pedigree of the two siblings with combined pituitary hormone deficiency; (B) the Propl gene mutation 301- 302delAG detected in the two siblings.

The male started treatment with GH when he was taller than the female (SDS: -2.59 vs.-3.79) and, although treated for a much shorter period, attained a better final height (SDS:+ 0.05 vs.- 0.28). This phenotypic variability is difficult to explain fully. A possible mechanism lies in the observation that, in Ames mice with Prop gene defects, a small number of somatotropes and thyrotropes are functional owing to clonal clusters of functioning cells [8]. In a study of Ames mice, GH secretion was detectable in 100 cells per pituitary gland [24]. Therefore, the variable delay in manifestation of GH deficiency in humans, even among those carrying the same molecular defect, might be compatible with variations in the number of somatotrope clones. Another possible explanation for the better outcome of the male proband is the fact that GH failure was manifested at the average age of pubertal initiation and GH treatment coincided with pubertal development when males are known to achieve their most accelerated linear growth. This pubertal growth spurt is more accelerated and more sustained in males than in females [25].

Patients with Prop1 mutations fail to enter puberty spontaneously, especially those homozygotes or compound heterozygotes whose mutation results in complete loss of function, while those with Prop1 mutations causing partial loss of function might present with spontaneous puberty and subsequent pubertal arrest. The patients of this study who were carrying the 301- 302delAG showed an absence of pubertal development and puberty had to be initiated by administration of sex steroids. In the male, hCG was added to the treatment in order to achieve testicular growth, essential for both psychological support of the patient and a better final result in future treatment for the restoration of fertility [26,27].

ACTH deficiency might be present later in life in some cases of CPHD due to Prop1 gene defects [8]. Cortisol response to insulin stimulation was normal in the patients of the present study. A progressive late-onset ACTH deficiency with the risk of shock due to acute corticosterone insufficiency has been reported only for CPHD patients carrying Prop1 mutations [19,29]. Phenotypic variability exists even between patients with the same 301-302delAG mutation. A patient with the 301-302delAG mutation had normal cortisol secretion at 8.8 years and at 16.6 years had developed partial cortisol deficiency, whereas another with the same molecular defect maintained normal cortisol secretion at 28.4 years [29]. Therefore, the patients of the present study need constant monitoring for delayed appearance of ACTH deficiency.

In conclusion, these two siblings of different sexes with CPHD carrying the 301-302delAG mutation in the Prop1 gene presented a variable phenotype characterized by GH, TSH, LH and FSH deficiency.

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NEOKLIS A. GEORGOPOULOS1, ILIAS KATSIKIS2, PETROS GIAMALIS1, VASILIKI KOIKA1, GEORGE ADONAKIS1, ANARGYROS KOURTIS2, GEORGE KOUROUNIS1, & DIMITRIOS PANIDIS2

1 Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Patras Medical School, Patras, Greece, and 2 Division of Endocrinology and Human Reproduction, second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Thessaloniki, Greece

(Received 15 June 2006; revised 20 September 2006; accepted 25 September 2006)

Correspondence: N. A. Georgopoulos, Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Patras Medical School, Rio-26500, Greece. Tel: 26 10 999835. Fax: 26 10 993854. E-mail: neoklisg@hol.gr

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