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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2177
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1909-1919
Copyright © 2007 by The Endocrine Society

Four Novel Mutations of the LHX3 Gene Cause Combined Pituitary Hormone Deficiencies with or without Limited Neck Rotation

Roland W. Pfaeffle, Jesse J. Savage, Chad S. Hunter, Christina Palme, Martina Ahlmann, Prasanna Kumar, Jaele Bellone, Eckhard Schoenau, Eckhard Korsch, Jürgen H. Brämswig, Heike M. Stobbe, Werner F. Blum and Simon J. Rhodes

University Children’s Hospital (R.W.P., C.P., H.M.S., W.F.B.), 04317 Leipzig, Germany; Department of Biology (J.J.S., C.S.H.), Indiana University-Purdue University, Indianapolis, Indiana 46202-5120; University Children’s Hospital (M.A., J.H.B.), D-48149 Münster, Germany; M. S. Ramaiah Medical College (P.K.), 560054 Bangalore, India; Children’s Hospital Regina Margherita (J.B.), 10126 Torino, Italy; University Children’s Hospital (E.S., E.K.), 50931 Cologne, Germany; Lilly Research Laboratories (W.F.B.), 22419 Bad Homburg, Germany; and Department of Cellular and Integrative Physiology (S.J.R.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Simon J. Rhodes, Ph.D., Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Medical Science Room 362A, 635 North Barnhill Drive, Indianapolis, Indiana 46202-5120. E-mail: srhodes{at}iupui.edu; or Roland Pfaeffle, M.D., University Children’s Hospital, Oststrasse 21-25, 04317 Leipzig, Germany. E-mail: rpfaeffle{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The Lhx3 LIM-homeodomain transcription factor gene is required for development of the pituitary and motoneurons in mice. Human LHX3 gene mutations have been reported in five subjects with a phenotype consisting of GH, prolactin, TSH, LH, and FSH deficiency; abnormal pituitary morphology; and limited neck rotation.

Objective: The objective of the study was to determine the frequency and nature of LHX3 mutations in patients with isolated GH deficiency or combined pituitary hormone deficiency (CPHD) and characterize the molecular consequences of mutations.

Design: The LHX3 sequence was determined. The biochemical properties of aberrant LHX3 proteins resulting from observed mutations were characterized using reporter gene and DNA binding experiments.

Patients: The study included 366 patients with isolated GH deficiency or CPHD.

Results: In seven patients with CPHD from four consanguineous pedigrees, four novel, recessive mutations were identified: a deletion of the entire gene (del/del), mutations causing truncated proteins (E173ter, W224ter), and a mutation causing a substitution in the homeodomain (A210V). The mutations were associated with diminished DNA binding and pituitary gene activation, consistent with observed hormone deficiencies. Whereas subjects with del/del, E173ter, and A210V mutations had limited neck rotation, patients with the W224ter mutation did not.

Conclusions: LHX3 mutations are a rare cause of CPHD involving deficiencies for GH, prolactin, TSH, and LH/FSH in all patients. Whereas most patients have a severe hormone deficiency manifesting after birth, milder forms can be observed, and limited neck rotation is not a universal feature of patients with LHX3 mutations. This study extends the known molecular defects and range of phenotypes found in LHX3-associated diseases.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
DURING PITUITARY GLAND development, the actions of transcription factors control the development of the hormone-producing cell types (reviewed in Refs. 1 and 2). Defects in transcription factor genes, including PIT1/POU1F1, PROP1, GLI2, HESX1, LHX3, and LHX4, are associated with combined pituitary hormone deficiency (CPHD) (3, 4). In general, hormonal insufficiencies of the affected individuals correspond to the expression patterns of the transcription factors within the cell types of the developing pituitary. Mutations in genes that are expressed early during development (e.g. HESX1) often cause variable clinical outcomes. Moreover, phenotypic differences are observed between individuals carrying a similar mutation within the same gene and also between mice with targeted mutations and human patients (5, 6, 7, 8, 9, 10, 11).

LHX3 is a LIM (Lin-11, Isl-1, and Mec-3)-homeobox gene expressed early during pituitary development (12, 13, 14, 15). The human LHX3 gene maps to chromosome 9 at 9q34.3 and has seven coding exons and six introns (16, 17). The LHX3 protein possesses two LIM domains involved in protein-protein interactions, a DNA binding homeodomain, and a carboxyl terminus that includes the major trans-activation domain (14, 18) (Fig. 1Go). In humans, three LHX3 isoforms are found: LHX3a and LHX3b, which have alternate amino termini, and a shorter protein, M2-LHX3 (18, 19). LHX3a, the most active isoform, can activate the promoters of pituitary genes, including the FSHß, {alpha}GSU, prolactin (PRL), TSHß, GnRH receptor, and PIT1 promoters (14, 19, 20, 21, 22).


Figure 1
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  		FIG. 1. A, Genomic organization of the human LHX3 gene. The boxes show the coding exons and the structure of the LHX3a mRNA with the coding regions for the LIM domains and the homeodomain (HD). The location of three observed mutations is indicated. A fourth mutation involves deletion of the gene. B, Predicted LHX3 proteins resulting from the mutations reported here. Zn, Zinc coordinated by the LIM domains; {alpha}1–3, predicted {alpha}-helices of the homeodomain. The carboxyl terminus contains the major transactivation domain (18 ). C, Radiolabeled wild-type (WT) and mutant LHX3a proteins expressed in vitro after PAGE. The relative molecular mass of reference proteins is given in kilodaltons.

 
In mice, Lhx3 is expressed as early as embryonic d 8.5 and is found in Rathke’s pouch and the closing neural tube around embryonic d 9.5 (12, 13, 14, 23, 24). Lhx3 is an early marker of anterior pituitary development and its continued expression suggests a role in the maintenance of the hormone-secreting cells. Homozygous Lhx3 knockout mice display an early arrest of pituitary development; lack the somatotrope, lactotrope, thyrotrope, and gonadotrope cell types; and die around birth (15, 24).

Homozygous recessive LHX3 mutations have been reported in five patients within three unrelated families (25, 26). Three siblings had a point mutation (LHX3a Y111C), which affects the second LIM domain, whereas one patient in another family had a 23-bp deletion, which caused a frameshift leading to a loss of the homeodomain. Functional analyses revealed loss of gene activation capacity of these mutant proteins (27, 28). A third class of LHX3 mutation involves a deletion in exon 2 causing a frame shift predicted to result in inactive peptides (25). These patients presented with a deficiency of all pituitary hormones but ACTH. In magnetic resonance imaging (MRI) views, some of the patients had small pituitaries, whereas others had enlarged glands. All patients showed elevated anteverted shoulders, leading to the clinical appearance of a stubby neck with severe limitation of cervical rotation. No abnormalities were visible at the cervical spine or the surrounding soft tissues. Therefore, it was suggested that the limited neck rotation may be due to abnormal innervation of the anterior neck muscles. Consistent with this, the transient expression of Lhx3 in the mouse spinal cord has been demonstrated to play a role in directing axon projections of spinal motoneurons to the ventral side of the neural tube (23, 29).

Because LHX3 is expressed early in pituitary development, we hypothesized that a variable phenotype exists in patients with LHX3 mutations. We screened a large cohort of patients with combinations of pituitary hormone deficiencies, with and without dysmorphic features, for the presence of LHX3 mutations. The objectives were to estimate the frequency of LHX3 gene mutations and establish correlations between specific mutations, the biochemical properties of the mutant proteins, and their phenotypic consequences. This information could help to identify those patients with CPHD who would benefit from DNA testing and also guide the clinician as to which hormone deficiencies can be expected in patients with LHX3 mutations.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Experimental subjects

We studied 295 patients with CPHD from 22 countries in Lilly’s Genetics and Neuroendocrinology of Short Stature International Study (GeNeSIS) program and 23 patients with CPHD followed up in our own clinic (318 patients from 302 pedigrees). All patients had GHD defined by stimulated GH levels of less than 10 ng/ml and at least one additional pituitary hormone deficiency indicated by a stimulatory test. (The frequency of the various combinations of hormone deficiencies in our patient sample is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.) Additionally, 48 patients (from 40 pedigrees) with isolated GH deficiency (IGHD) were studied to test the hypothesis that some LHX3 mutations might manifest as an incomplete form of pituitary insufficiency with only IGHD. Patients with IGHD had a maximal GH level of less than 10 ng/ml on stimulatory testing; 11 patients had complete GHD defined by stimulatory levels of less than 3 ng/ml.

After written informed consent was given, blood samples were collected from patients and, whenever possible, first-degree relatives. The study was approved by the local institutions’ ethics review boards according to the Declaration of Helsinki.

Screening for LHX3 mutations

LHX3 fragments were amplified from peripheral blood DNA in six fragments covering the entire coding and flanking intronic sequences of the gene (PCR primer sequences are published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Screening for sequence aberrations was performed by temperature modulated heteroduplex HPLC on a reverse-phase column using the WAVE-System (Transgenomics, Crewe, UK). Equal amounts of a patient’s PCR product and the corresponding fragment from an unaffected control subject were mixed and heat denatured. Possible heteroduplex formation was allowed to occur by gradually cooling the sample. Samples were analyzed on the WAVE-System and DNA fragments were detected by an UV detector. Temperature profiles for each fragment were optimized using the Wavemaker software (Transgenomics). Samples that showed an abnormal profile were analyzed by direct sequencing. PCR products were directly sequenced in duplicate using an ABI PRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA).

Cytogenetics

If the absence of amplified PCR fragments suggested complete deletion of the LHX3 gene, lymphocytes were isolated and cultivated in RPMI 1640 medium. Karyotyping was performed using standard procedures and Giemsa-staining. Fluorescence in situ hybridization (FISH) analysis of the subtelomeric region of chromosome 9 was performed using probe D9S235 (VYSIS). Short tandem repeat (STR) mapping was performed for patient C1 and unaffected parents at the LHX3 locus (138, 227, 917–138, 236, 776) using flanking marker D9S1826 (137, 588, 145–137, 588, 279), which is approximately 640 kb upstream of LHX3, marker D9S158 (138, 238, 885–138, 239, 107) located approximately 2 kb downstream of LHX3 and marker D9S905 (138, 995, 046–138, 995, 335) located approximately 760 kb downstream of LHX3. Analyses were performed using commercially available primers and an ABI Prism 377 genetic analyzer and ABI Genescan software.

Plasmid construction, transfection, and luciferase assays

LHX3a and LHX3b plasmids have been described (18, 19). A210/215V, E173/178ter, and W224/229ter mutations were introduced using the QuikChange kit (Stratagene, La Jolla, CA). Mouse pituitary GHFT1 cells and human embryonic kidney 293T cells were cultured and transfected as described (27). Luciferase activity was measured 48 h after transfection (27). All assay points were performed in triplicate. Total cell protein was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA), and luciferase activity was normalized to protein concentration.

Protein preparation and EMSA

LHX3 proteins were synthesized in vitro and analyzed as described (27). EMSAs were performed as described using in vitro-translated proteins and oligonucleotides (5'-gatcccagaaaattaattaattgtaa-3') representing a high-affinity LHX3 site (30).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Frequency of LHX3 mutations

We identified seven subjects in four families (1.9%) with LHX3 mutations from 366 patients with pituitary insufficiency representing 342 pedigrees. None of the 48 IGHD patients had LHX3 sequence variations other than polymorphisms present in the normal population. Excluding patients with IGHD, an LHX3 mutation was present in seven of 318 patients with CPHD (2.2%) and identified in four of 302 different pedigrees (1.3%).

For screening DNA fragments in patients with hypopituitarism, denaturing HPLC (dHPLC) proved to be a sensitive method to detect unknown mutations in amplified PCR fragments. In tests, it was able to identify 24 different known mutations within the POU1F1, PROP1, HESX1, GHRHR, and GH1 genes that we had previously detected in our laboratory by direct sequencing (data not shown). Its sensitivity under optimized conditions is reported to be between 95 and 100% (31). Of 2190 LHX3 fragments screened, 304 (14%) underwent resequencing due to an abnormal elution pattern in dHPLC: the majority of these represented known polymorphisms within exons 1a and 3. In 24 (8% of the resequenced fragments, 1.3% of all screened samples), no sequence abnormalities were detected and could be attributed to artifacts generated by the dHPLC screening procedure.

Patients with LHX3 mutations

Family A. Two children of a consanguineous Indian couple, a girl (patient A1) and a boy (A2), are homozygous for a mutation in exon 5. The mutation changes cytosine to thymine at position 629 of the LHX3a open reading frame (ORF) (position 643 of LHX3b), causing a conversion of alanine 210 to valine (LHX3a A210V; Fig. 1Go, A and B). The healthy parents and an unaffected brother are heterozygous for the A210V mutation (Fig. 2AGo). Patient A1 is the oldest of three children. Born at term with a birth weight of 3000 g, she experienced prolonged jaundice. Hypothyroidism was diagnosed at 1 month of age and she was briefly treated with T4, which was discontinued due to vomiting. Retrospectively from about 6 months of age, decreased height velocity was observed. She was finally referred to the endocrinologist at age 13.6 yr with a height of 82.5 cm [height SD score (SDS) –11] and a bone age of 2.7 yr. One month before presentation, she had experienced a generalized seizure while hypoglycemic with a blood glucose of 28 mg/dl. At presentation she had prominent eyes, frontal bossing, anteverted nares, and a depressed nasal bridge. Additionally, she has a short neck, elevated shoulders, and limited neck rotation restricted to 35° to both sides. MRI of the cervical spine showed loss of lordosis but otherwise normal anatomical structures (Fig. 3AGo). She was finally diagnosed with GH, TSH, and PRL) deficiency; gonadotrope function also is compromised because her LH/FSH response was low and she still remains prepubertal. Her morning cortisol level was 8 µg/dl at 0800 h and within the normal range (Table 1Go). MRI showed a slightly enlarged pituitary at normal location (6.6 mm height, 10 mm length, 12 mm width) with a volume of 396 mm3 (Table 2Go). According to the normative data for pituitary sizes in children (32, 33), both pituitary height and volume was high with respect to age (17 yr) and body height (117 cm) The most striking finding, however, was an abnormally high signal intensity of the anterior pituitary tissue on T1-weighted pictures in the absence of contrast enhancement (Fig. 3BGo). Treatment was initiated with recombinant GH and T4. After 11 months she had gained 13.5 cm in height and her bone age had increased to 8.5 yr (Fig. 4Go).


Figure 2
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  		FIG. 2. Pedigrees of families A, B, C, and D showing consanguinity of the parents (double lines). Genetic analyses were performed in parents and siblings except those indicated by {diamond}, who were not available. Two elder brothers of patient B1 had died postnatally. Representative DNA chromatograms are shown. Sequences of wild-type and mutated LHX3 genes are depicted for each of the point mutations. Filled symbols indicate homozygosity of the mutation; half-filled symbols indicate heterozygosity. Circles, female; squares, male.

 

Figure 3
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  		FIG. 3. A, Photographs of patients A1, A2, and C1 and cervical MRI of patient A1. B, T1-weighted section MRI scans of the brains of the indicated patients. A1 and A2 without gadolinium; B1, C1, D1, and D2 are after gadolinium. All images are sagittal except that for patient B1. The arrow indicates pituitary position.

 

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  		TABLE 1. Hormone profiles of patients with LHX3 mutations

 

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  		TABLE 2. Features of patients with LHX3 mutations

 

Figure 4
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  		FIG. 4. Growth curves of the affected patients. Dotted lines represent data before GH treatment; solid lines indicate growth after GH treatment.

 
Her youngest brother (A2) was asymptomatic at birth but showed early growth failure and similarly an apparent short neck and limited neck rotation. At referral he was 9.1 yr of age with a height of 78.5 cm (height SDS –8.7) and a bone age of 3 yr. He showed similar dysmorphic features to his sister. He has normally descended testes. Pituitary hormone testing revealed deficiency for GH and TSH. PRL was not determined and LH and FSH were still found in the prepubertal range (Table 1Go). Serum morning cortisol was low at 0800 h with 0.9 µg/dl and an ACTH test (1 µg iv) produced a blunted response to 2.0 µg/dl after 30 min and 0.7 µg/dl after 60 min. When MRI was performed at 14 yr of age, it revealed a pituitary with slightly increased pituitary height (height 6.6 mm, length 8.9 mm, width 10 mm). Calculated pituitary volume was 294 mm3, which is in the upper normal range using the normative data of Fink et al. (33). The anterior pituitary was found at normal location with an abnormally high signal intensity in T1-weighted pictures in the absence of contrast enhancement (Fig. 3BGo). On replacement with only GH (0.25 mg/kg/wk) and T4 (50 µg/d) but off cortisol replacement, the patient showed an impressive catch-up growth of 17 cm in the first 6 months of treatment (Fig. 4Go). This patient has not been treated with testosterone to date due to a retarded bone age.

Family B. The affected son (patient B1, Fig. 2BGo) of a consanguineous Moroccan couple showed homozygosity for a complex mutation in exon 3, in which the GC bases in position 287/288 of the LHX3a ORF were deleted and replaced by a 4-bp insertion (TCCT) (Fig. 1Go, A and B). This mutation shifts the ORF starting from codon 72 resulting in the introduction of 77 incorrect amino acids and a premature stop codon at position 173 (E173ter). Both parents are heterozygous for the mutation. The patient was delivered at term by cesarean section (3400 g; 48 cm). Two older brothers had died within the first 24 h after birth for unknown reasons. As a newborn he had experienced mild respiratory distress and showed signs of hypopituitarism, hypoglycemia, and hyponatremia. At the age of 4 months, he was evaluated due to muscular hypotonia and feeding problems. Length SDS was –3.8 and macroglossia, dry skin, short arms, a short neck, and a depressed nasal bridge also were observed. He has normally descended testes. Hormone testing revealed deficiency for GH, TSH, and PRL; LH and FSH levels were found at low levels (Table 1Go). Low morning cortisol levels were 4 µg/dl. Considering the history of the two elder brothers, replacement therapy with T4 and hydrocortisone was started and GH substitution was introduced 2 months later at age 6 months. Thereafter a rapid catch-up growth to the 75th height percentile could be observed within the first 18 months (Fig. 4Go). MRI showed a hypoplastic anterior pituitary in the normal location (Fig. 3Go). Pituitary volumetry could not be performed due to the limited quality of available pictures. Two younger siblings are healthy and growing normally.

Family C. The son (patient C1, Fig. 2CGo) of a consanguineous Algerian couple was found to be homozygous for a complete LHX3 gene deletion. Whereas repeated attempts to amplify LHX3 gene exon fragments were unsuccessful, amplification of other genes (POU1F1, PROP1, HESX1, LHX4, SHH) was unproblematic. Moreover, attempts to amplify additional LHX3 fragments spanning several exons failed. The patient exhibited a normal male karyotype by Giemsa staining. FISH analysis with the probe D9S235 showed no obvious structural abnormality in the subtelomeric region of chromosome 9q. PCR amplification of LHX3 fragments in both parents did not reveal any abnormalities. STR mapping was also performed (see Subjects and Methods). For the LHX3-flanking markers D9S182 and D9S905, no biallelic deletions could be detected in both parents and patient. There was no amplification of the marker D9S158 (located closest to LHX3) in the patient. Both parents displayed only one detectable allele, which is suggestive of hemi- or homozygosity. In face of the fact that all coding exons of LHX3 were not amplified by PCR in the patient, the STR results confirmed a biallelic deletion of LHX3 in the patient. The maximal length of the deletion therefore is between bases 137,588,279 and 138,995,046, which would constitute a microdeletion with a maximal length of 1.4 Mb.

The patient was delivered at term by vacuum extraction (birth weight 3250 g, birth length 46.6 cm). Shortly after birth he developed respiratory distress and hypoglycemia and was treated for suspected septicemia. He developed prolonged jaundice and frequent vomiting. Because of repeated hypoglycemic seizures, the patient was evaluated at the age of 2 months. Dysmorphic features were observed, including short arms and short neck with limited neck rotation (Table 2Go). He has normally descended testes. Hormone testing revealed hypothyroidism (free T4 < 0.05 ng/dl) with low serum TSH (0.03 µU/ml), low IGF-I (5.8 µg/liter), and low PRL level (<0.5 ng/ml). Serum cortisol was normal at 7.3 µg/liter. A pituitary hormone stimulation test with GHRH (1 µg/kg), CRH (1 µg/kg), and TRH (7 µg/kg) produced the maximal responses listed in Table 1Go, and it increased serum ACTH from 28.3 to 69 pg/ml, thereby excluding corticotrope insufficiency. MRI showed a severely hypoplastic anterior pituitary with almost no anterior pituitary tissue detectable and posterior pituitary at a normal location. After initiation of replacement therapy with GH and T4 at 2 months of age, the patient showed rapid catch-up growth, gaining 23 cm in 6 months (Fig. 4Go). His psychomotor development, however, remained severely retarded possibly due to the pronounced postnatal hypoglycemic episodes. At the age of 7 yr, x-ray and MRI of the spine showed a loss of cervical lordosis.

Family D. In three affected siblings of a consanguineous Lebanese couple, a sequence abnormality within exon 5 of LHX3 was identified. Guanine in position 672 of the LHX3a ORF was changed to adenine, thus introducing a premature stop codon predicted to cause loss of the carboxyl terminus (W224ter). Whereas the affected children were homozygous for this mutation, four of six unaffected siblings and the parents were heterozygous (Fig. 2DGo). Both the affected brother (D1) (birth weight 4000 g, length 53 cm) and his sister (D2) (birth weight 3500 g, length 51 cm) had an uneventful perinatal period with no reported episodes of hypoglycemia or prolonged jaundice. Both children grew persistently below the third height percentile after infancy. At the age of 9 and 8 yr, respectively, secondary hypothyroidism was diagnosed and T4 replacement was begun without a significant impact on height velocity. At 15 and 14 yr of age, they presented to the pediatric endocrinologist because of growth failure with height SDS of –5.6 and –5.2, respectively, and a bone age of 10.5 yr in both patients. Both children were prepubertal and were diagnosed with a combined pituitary hormone deficiency for GH, TSH, PRL, and LH/FSH by pituitary stimulation tests (Table 1Go). No further abnormalities or syndromic features were noted, especially no limited neck rotation. MRI showed pituitaries of normal size at the normal location (Fig. 3BGo). Additional replacement therapy with GH improved height velocity in the girl, less so in the boy due to inadequate compliance to therapy (Fig. 4Go). Puberty was induced at 16 yr 8 months in patient D2 with increasing doses of oral estrogens and at 17 yr 11 months in patient D1 with im testosterone. A younger sister of patients D1 and D2 (patient D3) also is homozygous for the affected allele. Less information is available for this patient, but she does have hormone deficiency and normal neck rotation.

Transcriptional properties of mutant LHX3 proteins

To study the molecular properties of the aberrant LHX3 proteins, expression vectors were constructed encoding wild-type and mutant proteins. In vitro transcription/translation analysis demonstrated that these vectors produced proteins of the predicted sizes (Fig. 1CGo; data not shown). LHX3 expression vectors were cotransfected with an {alpha}GSU reporter gene into 293T or pituitary GHFT1 cells. In both cell types, LHX3a activated the {alpha}GSU promoter, whereas the mutant LHX3 proteins did not (Fig. 5AGo). To test a distinct type of pituitary promoter, the PRL promoter/enhancer also was investigated. LHX3 alone is a weak activator of PRL transcription but in combination with PIT1, the effects are synergistic (14, 19). Alone or in combination with PIT1, the A210V, and E173ter LHX3a proteins did not activate the PRL promoter (Fig. 5BGo). There was some combined effect of the LHX3a W224ter protein with PIT1 on the PRL promoter in 293T cells but not in GHFT1 cells (which contain some endogenous PIT1).


Figure 5
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  		FIG. 5. Proteins encoded by mutated LHX3 genes display impaired transactivation properties. A, Expression vectors for wild-type (WT) and mutant LHX3 proteins were transiently cotransfected into heterologous human 293T kidney cells or mouse pituitary GHFT1 cells with a luciferase reporter gene under the control of the {alpha}-glycoprotein subunit ({alpha}GSU) promoter. Promoter activity was assayed by measuring luciferase activity 48 h after transfection. Negative controls (control) received equivalent amounts of empty expression vector plasmid. Activities are mean [light U per 10 sec/µg total protein] of triplicate assays ± SEM. A representative experiment of at least three experiments is depicted. B, Similar experiments to those depicted in A were performed using a PRL promoter/enhancer reporter gene with LHX3 and PIT1 expression vectors. C, DNA binding properties of proteins encoded by mutated LHX3 genes. EMSA experiments were performed using the indicated LHX3 proteins translated in vitro in rabbit reticulocyte lysates and radiolabeled probes representing the LHX3 consensus binding site (30 ). Unprogrammed lysates were used as negative controls (control). The LHX3/DNA complexes are denoted by the arrowhead. F, Free probe.

 
DNA binding properties of mutant proteins

Wild-type and mutant LHX3 proteins were generated in vitro and DNA binding was studied by EMSA analysis using radiolabeled oligonucleotides containing the LBC sequence, a specific, high affinity LHX3 binding site (30). Whereas the LHX3a E173ter mutant, which lacks the homeodomain, showed no DNA binding, there was remnant binding of the A210V protein (Fig. 5CGo), an observation consistent with the bulkier but similar chemical nature of the valine group. The LHX3a W224ter protein, which contains the homeodomain, showed slightly diminished binding. Similar observations were made for LHX3b and corresponding derivatives.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
By analyzing 366 patients, we demonstrated that LHX3 gene mutations are a rare cause of hypopituitarism. For comparison, in the same population, PROP1 mutations were more than 10 times as frequent, whereas HESX1 mutations had a similarly low prevalence. In contrast to HESX1 mutations, which can cause CPHD with variable penetrance even if patients are heterozygous (11), we did not identify any patients with CPHD who were heterozygous for LHX3 mutations. Subjects who were heterozygous for LHX3 mutations were clinically unaffected. These findings, consistent with the two prior reports (25, 26), suggest that CPHD due to LHX3 mutations is inherited in an autosomal recessive manner. By contrast, LHX4 gene mutations in patients described to date are heterozygous (34).

The scarcity of CPHD due to LHX3 mutations may be a consequence of several factors. First, all of the mutations identified in seven pedigrees to date are different, suggesting that there is no mutational hotspot. Second, it is possible that homozygous LHX3 mutations increase perinatal mortality as is observed in mice with null mutations in Lhx3 (24). This is suggested by the fact that two siblings of the patient with the E173ter mutation had died postnatally for unknown reasons. Moreover, the patient with the complete LHX3 gene deletion suffered from perinatal signs of hypopituitarism, which were initially interpreted as neonatal sepsis, a condition that could easily have ended fatally. In this patient who carries a microdeletion of 1.4 Mb or less, the phenotype could be modified by the possible absence of other genes located close to the LHX3 locus because we were not able to define the exact break points of this deletion by direct sequencing of the patient’s DNA. However, because the functions of most of the genes adjacent to LHX3 are yet unknown, their influence on the phenotype would remain speculative. Regardless, one can consider this patient to represent the phenotype of a null mutation of the human LHX3 gene.

Endocrine testing demonstrated deficiencies for GH, PRL, TSH, LH, and FSH in all affected patients with no differences between mutation types at the time of referral. This is consistent with the results of the functional tests of LHX3 activation capacities on pituitary genes. Because the endocrine tests were performed at different ages ranging from 2 months to 15 yr, it remains unclear whether hypopituitarism developed over time or whether it was fully present at birth. The latter may be suspected in the patients with A210V, E173ter, and del/del mutations due to the manifestation of hypopituitarism in the newborn period. By contrast, the patients with the W224ter mutation were not tested for hypopituitarism for approximately 9 yr because their growth seemed to parallel the third percentile for several years, suggesting some remnant pituitary function that diminished with time. This apparently less severe endocrine phenotype is consistent with the W224ter protein, which retains DNA binding capacity (Fig. 5CGo), having some residual function in the pituitary. Such a hypothetical deterioration of pituitary function in the patients with the W224ter mutation perhaps resembles the situation in patients with CPHD due to PROP1 gene mutations. They show a progressive decline of pituitary function (7), and the temporal pattern of this decline explains in part the variability of the clinical phenotype observed at the time of diagnosis. These differences in phenotype were not correlated with different genotypes. However, in adult life, all patients showed a deficiency of GH, PRL, TSH, LH, and FSH (7).

Of the patients with the early manifestation of hypopituitarism, patients A1 and C1 had cortisol levels in the lower normal range, and patients A2 and B1 had morning cortisol levels below the lower limit. In these two instances, this had prompted the physicians to initiate substitution therapy with cortisone temporarily in patient A2 and continuously in patient B1; however, in those patients in whom corticotrope cell function was determined with ACTH serum levels (patients B1, D1, and D2), these proved normal. Longitudinal observations of patients with PROP1 defects have shown that ACTH deficiency can manifest during adolescence or adulthood as a late complication of this form of CPHD (7, 35, 36), despite the fact that PROP1 is not expressed in the corticotrope cell. LHX3 is also not found in (rodent) corticotrope cells, and the development of ACTH deficiency would therefore be unexpected. However, future studies will determine whether a decline of corticotrope function occurs in patients with LHX3 gene mutations.

Besides the differences in endocrine findings, there were also differences between patients with different LHX3 mutations regarding pituitary morphology and the presence of dysmorphic features such as appearance of short neck and limited neck rotation. The patients with structural defects within the LIM domains and/or homeodomain show morphological abnormalities of their pituitary, including hypoplasia [del/del (this study), E173ter (this study), Y111C (26)], a hypointensity resembling a microadenoma (25), or pituitary enlargement with hyperintense MRI signal [A210V (this study), 23bp del (26)]. However, nothing can be said so far about temporal changes of pituitary morphology because no repeated imaging has been performed. By contrast, patients D1 and D2, with intact LIM domains and homeodomain, but lacking the carboxyl terminus (W224ter), showed normal pituitary morphology at 17 and 14 yr of age. It is tempting to link pituitary morphology to the sites of LHX3 mutations because there is no apparent discordance of this phenotypic sign among those siblings with the same mutation. Moreover, there is no apparent discordance between patients with mutations affecting the same functional domains.

Differences in pituitary size also are observed in patients and animal models with PROP1 mutations, independent of the site of mutation. Longitudinal studies have suggested a temporal pattern with enlargement followed by shrinking of the pituitary with several models proposed to explain this observation (7, 37, 38, 39, 40). Patients with LHX3 gene mutations and hypoplastic pituitaries reported so far are young, whereas those with enlarged pituitaries are more than 10 yr older. It is therefore unlikely that these patients follow the same pattern. Instead, it appears that in patients with LHX3 mutations, pituitary morphology is perhaps determined by the type of mutation.

LHX3 has an important role in the development of spinal cord motoneurons. This process requires formation of a multiprotein complex requiring the LIM domains and homeodomain of LHX3 (23, 29, 41). The carboxyl terminus of LHX3 contains the major transcriptional activation domains of the protein that allow signaling pathways to regulate LHX3 function and location (18, 42, 43). The LIM domains, however, also contain some transferable trans-activation function that could perhaps serve roles in the nervous system (18). The LIM domains also mediate interactions with partner proteins such as NLI and PIT1 (14, 27). The presence of intact LIMs and homeodomain may be sufficient for nervous system and some pituitary development in patients with the W224ter mutation, explaining the absence of limited neck rotation and abnormal pituitary morphology. Therefore, it appears that the location of the mutation within the LHX3 gene could influence these phenotypic features. The pituitary insufficiency in patients with the W224ter mutation, although manifesting later and with less severity, demonstrates the importance of the activation domain-containing carboxyl terminus in overall pituitary function. It is also possible that loss of the carboxyl terminus has secondary effects on the structure of the remaining protein such that interactions with other factors are altered or that this aberrant protein is subject to tissue-specific degradation (for example), resulting in an apparent dosage effect. Overall, however, we favor a model in which loss of the carboxyl terminal domains affects pituitary transcription functions but allows sufficient nervous system activity. Because we investigated only patients with pituitary abnormalities in our study, it is not clear whether a phenotype of limited neck rotation without any pituitary abnormality exists as a consequence of a LHX3 mutation.

We cannot examine LHX3 proteins in the pituitaries of these patients. It is possible that the premature termination codons in the E173ter and W224ter mRNAs results in targeting for nonsense-mediated decay, causing an absence of LHX3 proteins. However, there are exceptions to these rules (44), so the prediction for these cases is tentative. Furthermore, the delayed and different phenotype of the W224ter patients suggests that some LHX3 function might be retained in these subjects.

LHX3 mutations cause an autosomal recessive form of CPHD, creating a clinical phenotype of short stature, secondary hypothyroidism, and hypogonadism. Whereas most patients with LHX3 mutations will display limited neck rotation and abnormal pituitary morphology, certain mutations are not associated with these abnormalities as distinctive syndromic features. Although LHX3 mutations are rare, the identification of such mutations is important for clinical management of patients and genetic counseling and also is instrumental in understanding the mechanism of LHX3 in development.


   Acknowledgments
 
We are grateful to Drs. W. Heinitz and U. Froster for performing the FISH analysis and STR analysis and Drs. R. Maurer and P. Mellon for providing reagents. We thank Dr. Charmian Quigley and our colleagues for comments on the manuscript. We thank the patients for participation in this study.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD42024 and National Science Foundation Grant IBN 0131702 (to S.J.R.) and a grant from Lilly as part of the GeNeSIS (Genetics and Neuroendocrinology of Short Stature International Study) program (to R.W.P.). The GeNeSIS DNA substudy is part of a patient observational study and is sponsored by Eli Lilly International. J.J.S. was an Elizabeth Steele Creveling Memorial Scholar during this work.

Disclosure Statement: J.J.S., C.S.H., C.P., M.A., P.K., J.B., H.M.S., E.K., and S.J.R. have nothing to declare. R.W.P. and J.H.B. are on a Lilly advisory board and received lecture fees from Pfizer, Lilly, Ferring, and Novo Nordisk. E.S. is on a Lilly advisory board and received lecture fees from Pfizer, Lilly, Ipsen, and Novo Nordisk. W.F.B. is employed by Eli Lilly and Co. and has equity interests in that company.

First Published Online February 27, 2007

Abbreviations: CPHD, Combined pituitary hormone deficiency; dHPLC, denaturing HPLC; FISH, fluorescence in situ hybridization; GeNeSIS, Genetics and Neuroendocrinology of Short Stature International Study; IGHD, isolated GH deficiency; LIM, Lin-11, Isl-1, and Mec-3; MRI, magnetic resonance imaging; ORF, open reading frame; PRL, prolactin; SDS, SD score; STR, short tandem repeat.

Received October 5, 2006.

Accepted February 20, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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