Novel HESX1 Mutations Associated with a Life- Threatening Neonatal ...

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The Journal of Clinical Endocrinology & Metabolism 91(11):4528 – 4536 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0426

Novel HESX1 Mutations Associated with a LifeThreatening Neonatal Phenotype, Pituitary Aplasia, but Normally Located Posterior Pituitary and No Optic Nerve Abnormalities Marie-Laure Sobrier, Mohamad Maghnie, Marie-Pierre Vie´-Luton, Andrea Secco, Natascia di Iorgi, Renata Lorini, and Serge Amselem Institut National de la Sante´ et de la Recherche Me´dicale U654 Hoˆpital Henri-Mondor (M.-L.S., M.-P.V.-L., S.A.), 94000 Cre´teil, France; Department of Pediatrics (A.S.), University of Pavia, 27100 Pavia, Italy; and Department of Pediatrics (M.M., N.d.I., R.L.), Instituto di Ricovero e Cura a Carattere Scientifico G. Gaslini, University of Genova, 16147 Genova, Italy Context: Hesx1 is one of the earliest homeodomain transcription factors expressed during pituitary development. Very few HESX1 mutations have been identified in humans; although in those cases the disease phenotype shows considerable variability, all but one of the patients display an ectopic posterior pituitary and/or optic nerve abnormalities. Objective: The objectives of the study were to describe the complex phenotype associated with the panhypopituitarism of two unrelated Italian patients who, at birth, presented with hypoglycemic seizures and respiratory distress complicated by shock, in a familial context of neonatal death in one family and spontaneous miscarriage in both families and to identify the molecular basis of this unusual syndrome. Main Outcome Measures: Magnetic resonance imaging of the pituitary region, study of HESX1 gene and transcripts, and assessment of the ability of mutated HESX1 proteins to repress transcription were measured.

H

ESX1, A MEMBER of the paired-like class of homeobox genes, is first expressed in prospective forebrain tissue during mouse embryogenesis, but later, its expression is restricted to Rathke’s pouch, the primordium of the anterior pituitary gland (1, 2). This transcription factor contains two repressor domains, one located in the N-terminal region and the other in the homeodomain; the N-terminal eh1 motif recruits a corepressor, called Tle1, which is essential to Hesx1 function (3). In the mouse, Hesx1 and Tle1 are coexpressed in Rathke’s pouch between embryonic day (E)9.5 and E12.5 (3); their expression diminishes rapidly around E12.5, a developmental stage that coincides with a strong expression of Prop1 in the pouch and the subsequent appearance of several anterior pituitary cell types (3). Elegant in vitro and in vivo

First Published Online August 29, 2006 Abbreviations: E, Embryonic day; FT3, free T3; FT4, free T4; MRI, magnetic resonance imaging; rh, recombinant human; SDS, sd score; SOD, syndrome septooptic dysplasia; SSEP, somatosensory-evoked potential. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Results: Magnetic resonance imaging examination showed an anterior pituitary aplasia in a flat sella turcica and a normally located posterior pituitary in both patients. A constellation of extrapituitary developmental defects were found in the two patients, but without any optic nerve abnormalities. Sequencing of HESX1 exons and their flanking intronic regions revealed two different homozygous mutations. A frameshift (c.449_450delAC) was identified in one case, whereas the other patient carried a splice defect (c.357 ⫹ 2T⬎C) confirmed by the study of HESX1 transcripts. If translated, these mutations would lead to the synthesis of truncated proteins partly or entirely lacking the homeodomain, with no transcriptional repression, as shown by their inability to inhibit PROP1 activity. Conclusions: These observations reveal two novel HESX1 mutations in a so-far-undescribed disease phenotype characterized by a life-threatening neonatal condition associated with anterior pituitary aplasia, in the absence of ectopic posterior pituitary and optic nerve abnormalities, two features classically associated with HESX1 defects. (J Clin Endocrinol Metab 91: 4528 – 4536, 2006)

studies (3, 4) demonstrated the functional antagonism between Hesx1 and Prop1, a factor that belongs to the same family of paired-like homeodomain proteins. Hesx1 has been shown to function as a repressor of Prop1-mediated gene stimulation: Hesx1 or Prop1 homodimers, as well as Hesx1/ Prop1 heterodimers, are indeed able to recognize similar binding sites (3–5); in addition, the sequential overlapping temporal expression patterns of Hesx and Prop1 have been shown to be required for proper pituitary organogenesis (3). The targeted disruption of Hesx1 in the mouse results in variable anterior central nervous system defects that are highly reminiscent of the human syndrome septooptic dysplasia (SOD) (2). Hesx1⫺/⫺ mouse embryos exhibit either multiple oral ectoderm invaginations and/or cellular overproliferation of all pituitary cell types, or, in approximately 5% of cases, a complete lack of the pituitary gland (2, 6, 7). In 1998 Dattani et al. (2) found the first HESX1 molecular defect in humans; it was a missense mutation (p.R160C) identified in the homozygous state in two siblings with a SOD phenotype including agenesis of the corpus callosum and panhypopituitarism. To date, only eight mutations have been described in all four coding exons (2, 5, 8 –11). These

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Sobrier et al. • HESX1 Mutations and Pituitary Aplasia

J Clin Endocrinol Metab, November 2006, 91(11):4528 – 4536

mutations were identified in the homozygous (n ⫽ 3) or heterozygous (n ⫽ 5) state in patients presenting with isolated GH deficiency (5, 8) or combined pituitary hormone deficiency (2, 8 –11) and a constellation of extrapituitary abnormalities including midline brain defects, optic nerve hypoplasia, and coloboma. Only in two families is the posterior pituitary normally located (8, 11), whereas in all other cases it is ectopic (undescended). It was shown that, depending on the gene defect studied, the DNA binding activity of the mutant HESX1 protein was absent (9), decreased (2, 8), or increased (5), whereas in one case, the mutation impaired the ability of HESX1 to recruit the corepressor TLE1 (10). Recently an insertion of an Alu element within the HESX1 coding sequence, leading to the skipping of the corresponding exon, was identified in two siblings with pituitary aplasia (11); this was the first time that one of the numerous transcription factors involved in pituitary development was linked to this severe anomaly in the human. In the present study, we describe the complex phenotype associated with the panhypopituitarism of two unrelated patients who presented with a life-threatening neonatal condition in a familial context of neonatal death in one family and spontaneous miscarriage in both families. Both the severity of the disease phenotype and family histories prompted us to search for a germinal mutation in the early expressed gene HESX1. Patients and Methods Patients The patients or their parents provided their written informed consent to perform genetic studies. Both patients are females with panhypopituitarism and pituitary aplasia diagnosed in the neonatal period based on biochemical and neuroimaging findings. Their clinical phenotypes are described in details in Tables 1 and 2.

TABLE 1. Clinical phenotype of the two patients at the time of diagnosis Findings

Clinical and auxological parameters at birth Pregnancy course Gestational age at birth Delivery Birth weight Birth length Apgar score Neonatal period Respiratory distress Hypotonia Areflexia Hypothermia Severe hypoglycemia Seizures Hypocalcemia Diffuse peripheral edema Neonatal jaundice Neonatal sepsis

Family 1 Patient II2


Normal 41st wk ⫹ 3 d

Normal 40th wk

Induced labor

Cesarean section (for uterine inertia) 3.510 kg

3.350 kg 45 cm 7 at 1⬘, 10 at 5⬘

10 at 1⬘, 10 at 5⬘

⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹

⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺

⫹ ⫹

⫹ ⫺

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TABLE 2. Clinical phenotype of the two patients during follow-up Findings



⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹

⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺

⫹ ⫺ ⫺ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫺ ⫺ ⫹

⫺ ⫺ ⫹

⫹ ⫹ ⫺

Head and neck Microcephaly Macrocephaly Hypertelorism Antimongoloid fold of the eyelid Low base of the ears Large ears Saddle nose Ogival palate and dental malposition Macroglossy Lax skin at the nape of the neck Skeleton Large anterior fontanel Diastasis of sutura sagittalis Large posterior fontanel Bilateral congenital hip dysplasia Troncular obesity Flat feet Motility Neonatal poor spontaneous motility Persistent motor delay Swinging gait

Endocrinological investigations The diagnosis of GH deficiency was based on clinical criteria and biochemical findings of an extremely low GH response after arginine and/or glucagon tests. Pituitary-thyroid function was evaluated by measuring serum free T4 (FT4), free T3 (FT3), and TSH. ACTH deficiency was defined as morning serum cortisol concentration of less than 3.6 ␮g/dl (100 nmol/liter). Serum FSH and LH were measured before and 30, 60, and 120 min after the iv administration of 100 ␮g of GnRH in patient 1. Hypogonadism was confirmed by lack of puberty and no increase in serum FSH or LH in response to GnRH at 15 yr of age. All hormone measurements were carried out by means of standard RIAs. Endocrine characteristics are summarized in Table 3.

Electrophysiological and imaging investigations Additional investigations included standard electroencephalograms, head and hand x-rays, and magnetic resonance imaging (MRI) of the brain including the analysis of the pituitary region. Pituitary MRI was performed with spin echo T1-weighted images, followed by T2weighted imaging. Sagittal and coronal MRIs were obtained using 2- to 3-mm sections before and after gadolinium administration.

Mutation analysis of the HESX1 gene and other candidate genes Genomic DNA was isolated from blood samples obtained from each individual using standard techniques. The entire HESX1 gene (GenBank TABLE 3. Endocrine characteristics of the two patients Endocrine status at diagnosis

T4 (n.v. 116 –232 nmol/liter) FT4 (n.v. 10 –24 pmol/liter) FT3 (n.v. 2.8 – 6.5 pmol/liter) TSH (n.v. 0.4 – 4.0 mIU/liter) Morning cortisol (n.v. 248 – 496 nmol/liter) ACTH (n.v. 6.0 –57.0 ng/liter) Prolactin (n.v. 2.5–17.0 ␮g/liter) Peak GH after arginine test (n.v. ⬎ 10 ␮g/liter) Peak GH after glucagon test (n.v. ⬎ 10 ␮g/liter)



7.7 NE NE ⬍0.03 Undetectable

NE 6.4 0.2 ⬍0.03 ⬍55

0.9 1.8 1

4 ⬍0.2 NE

0.7

⬍.1

NE, Not evaluated; n.v., normal values.

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accession no. NM_003865) was amplified using the following set of primers, leading to a 1924-bp fragment: S1 (5⬘-CCCTTGTCAGCGGAGCTATA-3⬘, sense) and AS1 (5⬘-TGCAGGAAAGAAAACATCACATT-3⬘, antisense). The LHX3 and LHX4 genes were also analyzed after amplification of each coding exon, as previously described (12, 13). Each exon was sequenced using upstream and downstream primers (sequences posted online) according to the thermal cycle sequencing big dye terminator protocol (ABI Prism 310 genetic analyzer; PerkinElmer Applied Biosystems, Foster City, CA).

RT-PCR analysis Total RNAs were extracted from Epstein-Barr virus-transformed lymphocytes. A reverse transcription was performed with the ImProm-II reverse transcription system (Promega, Madison, WI) and random hexamer primers. The resulting cDNA templates were PCR amplified with primers S1 and AS1. Aliquots of RT-PCR products were separated by electrophoresis on a 1% agarose gel stained with ethidium bromide and were subsequently sequenced using the following set of primers: 1F (5⬘-AGCTGTTGCTCTGTGCAGACCACGA-3⬘, sense) and 4R (5⬘-TCATGCTCTGCAATTAGAAGATAATTTCAC-3⬘, antisense).

Plasmid constructs The PROP1 cDNA was amplified using human pituitary cDNA (CLONTECH, Palo Alto, CA) as template. The PROP1 cDNA as well as the wild-type and mutated HESX1 cDNAs obtained previously were subcloned into the expression vector pcDNA3 (Invitrogen, Cergy-Pontoise, France). The resulting expression vectors were designated pPROP1, pHESX1wt, pHESX1mut1 (carrying the c.449_450delAC deletion), and pHESX1mut2 (encoding a protein resulting from the c.357 ⫹ 2T⬎C mutation). Resulting constructs were confirmed by sequencing of the inserts and vector flanking fragments. The reporter construct containing six PIII sequences upstream of a luciferase gene (pPIIILuc) was a gift from M. Dattani (Institute of Child Health, London, UK).

Cell culture and transfection HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in DMEM medium (Invitrogen) containing 10% fetal calf serum at 37 C. All transfections were performed at 60% confluence in 6-well culture plates by the Lipofectamine-Plus method (Invitrogen) in OptiMEM according to the manufacturer’s standard protocol.

Luciferase activity assays Cells were transfected with 100 ng of the pPIIILuc vector together with either empty pcDNA3 expression vector and/or the various PROP1- and HESX1-derived pcDNA3 constructs; the total amount of DNA transfected per well was normalized to 200 ng by addition of the appropriate amount of empty expression vector. The cell extracts were prepared and assayed for luciferase activity using the Promega assay system. Luciferase activity was normalized to protein concentration, which was measured using the Coomassie Plus protein assay reagent kit (Pierce, Rockford, IL). Each transfection experiment (in triplicate) was performed independently at least three times.

Results Patient phenotypes

Family 1, patient II2. The clinical phenotypic features of this patient at the time of diagnosis and during follow-up are summarized in Tables 1 and 2. Patient II2 (Fig. 1A) is an Italian girl born about 10 d postterm by induced labor at the end of a normal pregnancy. Her weight at birth was 3.350 kg, and her length was 45 cm. She has one older sister, with no relevant clinical problems, and had two younger siblings: one male, stillborn, diagnosed postmortem with pituitary aplasia (individual II3 in Fig. 2A); one female with pituitary aplasia who died at 26 d after severe hypoglycemia and

FIG. 1. Craniofacial phenotype of the two probands. A, Patient II2 from family 1 at 10 months (left) and 8 yr (right). B, Patient II3 at 5 months (left) and 4 yr (right).

seizures complicated by sepsis (individual II5 in Fig. 2A). One spontaneous abortion was noticed in one parental lineage (Fig. 2A). The patient’s parents, who are not known to be related, come from the same small village. At 15 h of life, patient II2 presented with cyanosis, hypotonia, areflexia, hypothermia, diffuse peripheral edema, severe hypoglycemia leading to systemic collapse necessitating resuscitation, and jaundice requiring full blood exchange at 4 and 8 d of age, when she also presented diffuse internal and external bleeding and was diagnosed as having diffuse intravascular coagulation, possibly secondary to sepsis (she had enteritis with stool positive for Salmonella and Pseudomonas). Immediately from her first hours of life, there were severe electrolyte abnormalities, particularly hypocalcemia, which required constant correction with iv calcium and vitamin D supplementation. Bilateral congenital hip dysplasia, slightly more relevant on the left side, was also observed on close clinical examination and confirmed by hip x-rays, repeated throughout the years (data not shown). Clinical phenotype included saddle nose, low base of the ears, antimongoloid fold of the eyelid, lax skin at the nape of the neck, ogivale palate, and microcephaly (Fig. 2C, right panel). Later on, the patient presented dental malposition, harmonious short stature with slight troncular obesity, swinging gait and mental retardation. In the first months of life, the baby suffered from recurrent episodes of hypoglycemia, convulsions, and recurrent skin,



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FIG. 2. Family history and main growth-related data of the patient from family 1. A, Genealogical tree of family 1. Filled symbol, affected; empty, unaffected. B, Sagittal T1-weighted MRI before (a) and after (b) gadolinium showing posterior pituitary hyperintensity (arrow) at the distal end of a slightly and homogeneously enlarged pituitary stalk, narrow anterior pituitary, and flat sella turcica (arrowhead). The corpus callosum is thin (double arrows). A Chiari type I malformation is shown by an asterisk. C, Growth pattern of patient II2: height and head circumference curves; duration of different endocrine treatments are shown by solid horizontal bars.

ear, and lower airway infections; one of the latter led to lung collapse and respiratory arrest requiring intensive care treatment for 10 d at 3 months of age. At time of presentation, the suspected diagnosis, based on clinical presentation and severe hypoglycemia, was panhypopituitarism. Laboratory results evidenced absent thyroid function (central hypothyroidism), total GH deficiency, and cortisol deficiency (Table 3). The karyotype was normal, as were subtelomeric analyses. At 3 months of age, immune work-up revealed severe deficiency of cellular immunity, whereas humoral immunity was normal. However, at 9 months, immune function reevaluation showed normal cellular and humoral immunity. The results of electrophysiological and imaging investigations, which are summarized in Table 4, showed diffuse unspecific alterations in brain electric activity and a small head presenting dolichocephaly with symmetric thickening of the inner table of the frontal bone compatible with intense hyperostosis, whereas hand x-rays revealed a stubby appearance of the distal phalanx of the III finger, which was laterally deviated. Compensated hydrocephalus was confirmed by ultrasonography. At 5.2 yr of age, a decompression of hypertonic hydrocephalus was performed by ventricularperitoneal derivation; the derivation tube was replaced at 11 and 26 yr of age. Replacement therapy with l-thyroxine and hydrocortisone was started at 1 and 3 months of age, respectively.

Treatment with pituitary human GH (Grorm; Serono, Geneva, CH) was also begun at 7 months of age, three times weekly im (11 IU/m2䡠wk) until the age of 6 yr. Growth velocity during this period was about 6 cm/yr, slowly decreasing up to a growth arrest between 6 and 11 yr leading to GH therapy withdrawal (Fig. 2C, left panel). Replacement therapy with recombinant human (rh)GH at a dose of between 15 and 18 IU/m2䡠wk sc was reintroduced at 11 yr, with an increase of height velocity up to 8 cm/yr. In 1999 (at 22 yr of age), the patient started rhGH therapy at adult dosage (0.5–1 mg/d sc). Ultrasonography was compatible with a prepubertal uterus. Transdermal 17 ␤-estradiol patches with medroxyprogesterone acetate (5–10 mg, 12th to 21st d) were started at 16 and 17 yr of age, respectively, with regular menses. Bone densitometry showed generalized (vertebrae, hands, hips) reduction in mineral bone density for age and sex (Table 4). MRI examinations, performed over the years, showed normal posterior pituitary gland, anterior pituitary gland visible as a thread-like formation within the sella turcica after iv contrast medium, pituitary stalk of enlarged width, particularly remarkable compared with the size of both the pituitary gland and the sella turcica, and Chiari I malformation (Fig. 2B and Table 4). At age 26 yr, the patient underwent surgical application of a left hip prosthesis for congenital hip dysplasia. At the most

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TABLE 4. Results of electrophysiological and imaging investigations in the two patients Findings

Electrophysiological studies Slow waves at electroencephalogram Altered SSEPs Bone densitometry Generalized (vertebrae, hands, hips) decreased mineral bone density for age and sex Radiological findings Dolicocephaly Absence of proximal tibial epiphysis at birth Hand x-ray abnormalitiesa MRI Posterior pituitary gland/normal location Anterior pituitary gland aplasia Flat sella turcica Normal pituitary stalk Thin corpus callosum Hydrocephalus Cerebral atrophy Chiari I malformation



⫹ NE

⫹ ⫹

⫹

⫺

⫹ ⫹

⫺ ⫹

⫹

⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹

a Hand x-ray abnormalities were stubby and laterally deviated distal phalanx of the III finger.

recent control, her adult height was 140.5 cm [⫺3.6 sd score (SDS)], her weight was 52.6 kg and BMI was 26.6 (⫹1.4 SDS). Family 2, patient II3. The clinical phenotypic features of this patient (Fig. 1B) at the time of diagnosis and during follow up are summarized in Tables 1 and 2. The propositus (individual II3 in Fig. 3A) is an Italian girl born at term of a normal gestation by cesarean section for uterine inertia. Her birth weight was 3.510 kg, her length was 49 cm, and Apgar

index was 10 at 1⬘ and 5⬘. She is the second of two daughters born to parents originating from the same region; her sister and parents have no relevant clinical problems. Between the two pregnancies, the mother had a spontaneous miscarriage at the fifth week of gestational age. Spontaneous miscarriages had also occurred in both parental lineages (Fig. 3A). During the first day of life, the patient presented respiratory distress with a crisis of generalized cyanosis and hypoglycemic seizures. She was transferred to the intensive care neonatal unit. Objective findings at admission were jaundice; poor spontaneous motility; large anterior and posterior fontanels with diastasis of sutura sagittalis; and several morphological abnormalities, such as apparently short inferior limbs, hypertelorism, saddle nose, low base of the ears, and macroglossa. The clinical presentation together with severe hypoglycemia led us to suspect congenital panhypopituitarism. She was therefore treated with parenteral glucose infusion, hydrocortisone, and l-thyroxine, which was followed by dramatic improvement. Assessment of thyroid function was compatible with central hypothyroidism (low FT3 and FT4 with nondetectable TSH). Findings of nondetectable morning cortisol, ACTH, and prolactin levels well below the range of normality and peak GH response after glucagon test of 0.1 ng/ml all suggested a diagnosis of multiple pituitary hormone deficiencies (Table 3). Consequently, long-term replacement therapy with l-thyroxine, hydrocortisone, and recombinant GH (18 –20 IU/m2䡠wk sc) was started and progressively adjusted as necessary. Furthermore, GH retesting in young adulthood confirmed nonmeasurable serum GH. Brain and pituitary MRI showed no central nervous system abnormalities, although complete anterior pituitary aplasia with normally

FIG. 3. Family history and main growth-related data of the patient from family 2. A, Genealogical tree of family 2. Filled symbol, affected; empty, unaffected. B, Sagittal T1-weighted MRI performed in the neonatal period before (a) and after (b) gadolinium showing posterior pituitary hyperintensity (arrow) at the distal end of the pituitary stalk, anterior pituitary agenesis and flat sella turcica (arrowhead). C, Growth pattern of patient II3: height and head circumference curves; duration of the different endocrine treatments are shown by solid horizontal bars.


located posterior pituitary and normal pituitary stalk was observed (Fig. 3B and Table 4). At the age of 8 yr, the girl underwent orthopedic surgical intervention for correction of bilateral flat foot. She has presented delays in motor development including slow gait and voluntary body movements up to the most recent follow-up at 10 yr of age. At this time, neurological examination was normal but inferior-limb somatosensory-evoked potentials (SSEPs) were altered. The patient has been attending school with satisfactory results. Her current rate of growth with GH at the dose of 0.027 mg/d is rather advanced (genetic height potential, ⫺1 SDS) with a height of ⫹1.6 SDS (Fig. 3C) and weight more than ⫹2 SDS. A recent brain and spinal chord MRI performed in 2004 confirmed pituitary aplasia and revealed the existence of Chiari I malformation. Comparisons between the clinical phenotype of the two patients, both at the time of diagnosis and during follow-up, and between endocrine findings and electrophysiological and imaging investigations are shown in Tables 1– 4. HESX1 mutation detection

DNA samples from several members of these two unrelated families were screened for mutations by direct sequencing, using HESX1-specific primers that amplified all the coding regions and intron-exon boundaries. These analyses revealed that the patient from family 1 (individual II2) carried a homozygous deletion of 2 bp in exon 3 (c.449_450delAC) (Fig. 4A). Segregation analysis of the mutant HESX1 allele revealed that her two parents (individuals I1 and I2) and her older sister (II1), who have a normal phenotype, carried the deletion in the heterozygous state (data not shown). In family 2, patient II3 was found to bear a homozygous T-to-C transition at position ⫹2 of exon 2 (c.357 ⫹ 2T⬎C), involving the invariant dinucleotide of the splice-donor site of intron 2 (Fig. 5A). The two healthy parents (I1 and I2) were shown to carry the same sequence variation in the heterozygous state, whereas the older sister (II1) inherited two normal HESX1 alleles (data not shown). The anterior pituitary aplasia documented in two patients also prompted us to screen for mutations two other early expressed genes, LHX3 and LHX4, whose targeted disruption in mice results in severe anterior pituitary hypoplasia

FIG. 4. Molecular data in family 1. A, Electrophoregrams of the normal and mutated HESX1 gene sequences showing a homozygous deletion involving two nucleotides of exon 3 (horizontal black line) at position 449 – 450 in patient II2. B, RT-PCR amplification of the HESX1 cDNA obtained from lymphocyte RNAs of a control (C) and patient II2 (top). Schematic representation of the transcripts corresponding to the normal full-length HESX1 transcript amplified from a control sample (1) and to the 2-bp deleted transcript amplified from the patient’s sample (2) with the modified reading frame shown by a hatched area and the premature stop codon at position 167 (bottom). The homeodomain (HD) is depicted by a dotted red line.


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(14, 15). No mutation was found in LHX4; however, a heterozygous sequence variation was identified in LHX3 in the patient from family 2 (c.935G⬎C) as well as in her healthy father and sister (individual II1). This sequence change would result in the replacement of Arginine 312 by a Proline residue. This substitution, found in several family members (affected or unaffected), involves a residue located in the C-terminal part of the protein, downstream of the homeodomain, which is not invariant throughout evolution, thereby suggesting that it could represent a benign polymorphism. In keeping with this hypothesis, we did not observe any effect of this variation on the transcriptional capability of the corresponding mutated LHX3 protein, as assessed on different target promoters (i.e. those from ␣GSU and ␤TSH) (data not shown). Consequences of the identified HESX1 sequence changes on the processing of transcripts

To determine the consequences of the two HESX1 molecular defects at the RNA level, RNA from Epstein-Barr virustransformed lymphocytes of the patients, their parents, and a control were extracted and submitted to RT-PCR, using primers flanking the entire HESX1 coding sequence. For the control, a product of expected size (762 bp) was amplified (Figs. 4B and 5B); sequencing of this product indeed showed that it results from a normal splicing of all intronic sequences between exons 1 and 4. The RT-PCR performed with the HESX1 cDNA of the patient from family 1 (individual II2) generated a product of size similar to that obtained from a control sample, but after sequencing, the 2-bp deletion was indeed detected in exon 3 so that the cDNA product is 760 bp long (Fig. 4B). This frameshift mutation would lead to a truncated HESX1 protein, deleting 30% of the C-terminal part of the homeodomain and all the following residues (Fig. 4B). The same experiment performed with the HESX1 cDNA of the patient from family 2 generated a smaller molecular species (562 bp), whereas a product of 762 bp corresponding to normal alleles was amplified from the sister’s (II1) cDNA and both the 762- and 562-bp products were amplified from the father’s (I1) and mother’s (I2) cDNAs (Fig. 5B). Sequencing of the smaller isoform revealed that it corresponds to a

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FIG. 5. Molecular data in family 2. A, Electrophoregrams of the normal and mutated HESX1 gene sequences showing, in patient II3, a homozygous T-to-C transition (vertical arrow) in the splice donor site of exon 2; the vertical line represents the exon 2-intron 2 junction. B, Genotype status of family members. Filled symbol, homozygous for two mutated alleles; half-filled, heterozygous; empty, homozygous for two normal alleles. RT-PCR amplification of the HESX1 cDNA obtained from lymphocyte RNAs of a control (C), the healthy parents (I1 and I2) and sister (II1), and the patient (II3) (top). Schematic representation of the transcripts corresponding to the normal full-length HESX1 transcript amplified from a control (1) and to the 562-bp exon 2-deleted transcript amplified from the patient’s sample (2) (bottom). The premature stop codon at position 54 is shown. The homeodomain (HD) is depicted by a dotted red line.

HESX1 transcript missing exon 2. If translated, this abnormal transcript would generate a protein lacking 70% of the protein, including the entire homeodomain, because of a premature stop codon at the beginning of the remaining exon 3 (Fig. 5B). Assessment of the ability of the two mutated HESX1 proteins to repress transcription

As mentioned earlier, HESX1 has been shown to function as a repressor of PROP1-mediated gene stimulation. To assess the ability of these two mutated HESX1 proteins to repress transcription, we therefore expressed them, in HEK293 cells, together with PROP1, in the presence of the pPIIILuc plasmid that contains consensus PIII target sites common to PROP1 and HESX1; as a control, the same experiment was performed with the plasmid encoding the normal HESX1 protein (pHESX1wt) (Fig. 6). As reported previously (4, 5), transfection of pHESX1wt alone had minimal effect on the PIII sequence, whereas transfection of pPROP1 resulted in a 5- to 7-fold stimulation of luciferase activity. As expected, cotransfection of pHESX1wt with pPROP1 re-

FIG. 6. Functional consequences of the two identified HESX1 molecular defects. HEK293 cells were transfected with the pPIIILuc plasmid in presence of the empty expression vector pcDNA3, pPROP1, pHESX1wt, pPROP1 and pHESX1wt, pPROP1 and pHESX1mut1, or pPROP1 and pHESXmut2. One representative experiment (done in triplicate) of three independent experiments is shown.

sulted in an inhibition of the PROP1-dependent activation of the PIII sequence. However, as shown by the cotransfection of pHESXmut1 or pHESXmut2 with pPROP1, such repression activity was lost in mutated HESX1 proteins. These data therefore clearly demonstrate the deleterious effect of the two identified mutations on HESX1 function. Discussion

This study identified two novel HESX1 mutations in patients presenting with an unusual disease phenotype characterized by a life-threatening neonatal condition associated with anterior pituitary aplasia, in the absence of ectopic posterior pituitary and optic nerve abnormalities. Given both the nature and the location of the identified molecular defects, the functional consequences of these mutations are expected to be highly deleterious; this was confirmed by in vitro means, the two mutated HESX1 proteins having lost their ability to inhibit PROP1 activity. Such defects may therefore have severe consequences on the delicate balance of the transcription factor molecular cascades required for the proper development of the pituitary region. The mutations so far identified in transcription factors involved in pituitary development (e.g. PIT1, PROP1, LHX3 and LHX4, and SOX3) are rare (16); this is especially the case for the factors expressed at early developmental stages, like HESX1 (with this study the number now totals 10), LHX3, LHX4, and SOX3. Such scarcity may be explained, at least in part, by the fact that mutations in early expressed genes are usually associated with a severe phenotype that could even be lethal (17). In keeping with this observation, it is striking to note that spontaneous miscarriages have occurred in our two families in which segregates a severe HESX1 molecular defect, with a rate of 12% in family 1 and 33% in family 2, whereas reported rates of clinically recognizable spontaneous abortions range from 12 to 20% in normal populations, depending on the study and examined population (18 –20). Nevertheless, given the small size of our sample, such comparisons should be taken cautiously. Noteworthy, in utero or neonatal death has also been described in a mouse Hesx1 knockout model (2). The most severe phenotype in those mice comprised decreased head size, short nose, and variable


anterior central nervous system defects; moreover, the mice died shortly after birth and all homozygous mutants were abnormal at birth. Altogether these phenotypic features are highly reminiscent of the severe phenotype presented by our two patients. Such adrenal crisis as presentation of hypopituitarism is, however, not typical. Indeed, adrenal crisis is not the main feature of hypopituitarism, except in case of absent anterior pituitary gland. Neonatal hypopituitarism associated with pituitary aplasia is life threatening because of the risk of sudden death from hypoglycemia and cortisol deficiency. Besides, adrenal hypoplasia or aplasia has been reported at autopsy in almost all infants with anterior pituitary agenesis, suggesting that this condition requires prompt initiation of diagnostic tests, correction of hypoglycemia, and replacement therapy without undue delay (21, 22). Noteworthy, Tajima et al. (9) identified a heterozygous 2-bp insertion in HESX1 leading to the loss of the homeodomain and thus generating a protein similar to that expected from the skipping of exon 2 herein described. However, in that case, the disease phenotype was dominant and less severe than in our patient who carried a homozygous HESX1 defect and who was born to healthy heterozygous parents. Such phenotypic differences among individuals carrying similar defects in the heterozygous state may reflect substantial differences in the genetic background and the existence of modifier genes that remain to be identified. Compared with most patients so far described with a HESX1 defect, the phenotype of our two patients is unusual in that, despite its particular severity (i.e. pituitary aplasia revealed by life-threatening neonatal panhypopituitarism), it includes a normally located posterior pituitary and no optic anomalies. Whereas these two unrelated patients shared these characteristics, they did not, however, display the same extrapituitary abnormalities. The MRI data in fact confirm the role of HESX1 in pituitary organogenesis and in the development of the corpus callosum as well as unveil a so-far-unsuspected role of this protein in the proper development of the cerebellum. In this regard, the Chiari I malformation diagnosed in the two patients recalls the phenotype associated with a mutation in another transcription factor expressed early, namely LHX4 (13), suggesting a possible functional link between HESX1 and LHX4 for the development of the cerebellum. On the other hand, the motor delay observed in the patient with altered inferior-limb SSEPs is rather intriguing and brings to mind the limited neck rotation described in patients with a LHX3 mutation (12) and that most probably results from a LHX3-dependent neurological defect that is not rescued by the closely related LIM homeodomain-containing protein LHX4 (23). Noteworthy, such potential links between LIM homeodomain-containing proteins and HESX1 have just recently been documented in mice (24). The other clinical features described in our patients, including facial characteristics, head circumference, and skeletal abnormalities as well as the response to rhGH, further document the high degree of variability of phenotypes associated with HESX1 anomalies; however, given the very probable consanguinity in the two families, the participation of additional unrecognized mutation(s) in this complex disease phenotype cannot be excluded.


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These observations, which broaden the clinical pictures resulting from HESX1 mutations, underscore the pleiotropic effects of this early expressed transcription factor during development. They also disclose the necessity to look for HESX1 defects in patients with pituitary aplasia and a normally located posterior pituitary in the absence of SOD. Such studies, which might contribute to deciphering the molecular bases of combined pituitary hormone deficiency, should be essential for the best management of these disorders, including accurate genetic counseling of affected families. Acknowledgments We are indebted to the children and their parents for agreeing to participate in this study. We thank M. Dattani (Institute of Child Health, London, UK) for the gift of the pPIIILuc plasmid. Received February 23, 2006. Accepted August 8, 2006. Address all correspondence and requests for reprints to: Serge Amselem, M.D., Ph.D., Institut National de la Sante´ et de la Recherche Me´dicale, U654, Hoˆpital Henri-Mondor, Cre´teil F-94010, France. E-mail: [email protected]. This work was supported by the Institut National de la Sante´ et de la Recherche Me´dicale and grants from the Assistance Publique-Hoˆpitaux de Paris (CRC96085, PHRC 2003 25/2003).

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