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CLINICAL CASE SEMINAR |
Pediatric Endocrinology Division (A.P.S.B., S.T.) and Pediatric Neurology Division (S.P.), Infant’s and Children’s Hospital of Brooklyn at Maimonides, Brooklyn, New York 11219; Department of Biology (C.S.H., J.J.S.), Indiana University-Purdue University, Indianapolis, Indiana 46202; Pediatric Endocrinology Division (H.A.), Saint Barnabas Medical Center, Livingston, New Jersey 07039; and Department of Pediatrics (E.C.W.), Section of Endocrinology and Diabetology, 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: Svetlana Ten, M.D., 977 48th Street, Brooklyn, New York 11219. E-mail: tenlana{at}aol.com.
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Abstract |
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Objective: We report a patient presenting a unique phenotype associated with a novel mutation in the LHX3 gene.
Patient: We report a 6-yr, 9-month-old boy born from a consanguineous relationship who presented shortly after birth with cyanosis, feeding difficulty, persistent jaundice, micropenis, and poor weight gain and growth rate. Laboratory data, including an undetectable TSH, low free T4, low IGF-I and IGF binding protein-3, prolactin deficiency, and LH and FSH deficiency were consistent with hypopituitarism. A rigid cervical spine leading to limited head rotation was noticed on follow-up examination. Magnetic resonance imaging revealed an apparently structurally normal cervical spine and a postcontrast hypointense lesion in the anterior pituitary.
Results: Analysis of the LHX3 gene revealed homozygosity for a novel single-base-pair deletion in exon 2. This mutation leads to a frame shift predicted to result in the production of short, inactive LHX3 proteins. The results of in vitro translation experiments are consistent with this prediction. The parents of the patients are heterozygotes, indicating a recessive mode of action for the deletion allele.
Conclusions: The presence of a hypointense pituitary lesion and other clinical findings broadens the phenotype associated with LHX3 gene mutation.
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Introduction |
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TopAbstractIntroductionPatient and MethodsResultsDiscussionReferences |
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Several LIM-homeodomain class transcription factors, including ISL1, LHX3, and LHX4, play critical roles in pituitary and nervous system development (7). These regulatory proteins have two LIM domains in the amino terminus that are involved in protein/protein interactions and a centrally located homeodomain that mediates interactions with DNA recognition sequences in target genes (7). The human LHX3 gene contains seven coding exons and six introns on chromosome 9q34 and encodes three protein isoforms of the LHX3 transcription factor (8, 9, 10). The three protein isoforms are LHX3a and LHX3b, which both contain the LIM domains and homeodomain but have different amino termini, and a shorter isoform (M2-LHX3) that lacks the LIM domains but retains some gene regulatory activities (8, 9, 10). In mice, the orthologous gene, Lhx3, is expressed in the embryonic nervous system and in the developing and mature pituitary (11, 12, 13). Transgenic mice with a homozygous deletion of Lhx3 display incomplete pituitary development, aberrant motor neuron specification, and perinatal death (14, 15, 16). In these animals, some ACTH is detectable, indicating at least partial corticotroph cell function, but the characteristic hormones of the other anterior pituitary cells are not found, indicating that Lhx3 is required for development of four of the five specialized cell types (16). Molecular studies have demonstrated that LHX3 can activate pituitary expressed genes, such as those encoding the 
-glycoprotein subunit (GSU), PRL, FSHß, TSHß, the GnRH receptor, and the Pit-1 transcription factor (9, 10, 13, 17, 18, 19).
To date, only one report has described molecular defects in LHX3 associated with a complex combined pituitary hormone deficiency (CPHD) syndrome (20). Two recessive mutations were reported: a missense mutation altering a single, conserved amino acid in the LIM domains (Y111C in the LHX3a protein) and a 23-bp deletion predicted to result in the translation of a truncated protein (20). The patients were from two unrelated, consanguineous families that presented with GH, TSH, LH, FSH, and PRL deficiencies and a short, rigid cervical spine with limited head rotation. Magnetic resonance imaging (MRI) demonstrated small anterior pituitaries in two patients, whereas another had an enlarged pituitary gland (20). These mutations are predicted to lead to the production of proteins that are ineffective in trans-activation of pituitary hormone gene promoters such as GSU and PRL (21, 22).
In this study, we describe a patient with a novel LHX3 gene mutation, CPHD with neck rigidity, a hypointense pituitary lesion radiologically consistent with a microadenoma, and other features including possible focal amyotrophy and speech delay. A homozygous single-base-pair deletion was found in exon II of LHX3. This mutation is predicted to result in the production of nonfunctional protein(s). The patient’s parents are cousins and are heterozygous for the mutation, consistent with a recessive mode of inheritance.
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Patient and Methods |
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A CRH stimulation test was performed with 1 µg/kg of ovine CRH as previously described (23). Serum cortisol was analyzed by HPLC tandem mass spectrometry method. ACTH was measured by the immunochemiluminometric assay (ICMA) method; LH and FSH were measured by an immunochemiluminometric assay method. IGF-I and IGF binding protein-3 (IGFBP-3) were measured with blocking RIA after acid/alcohol extraction and RIA in dilute serum methods (Esoterix, Calabasas Hills, CA).
Diagnostic procedures to detect mutations in LHX3
After consent, peripheral blood was collected in a green-top heparin vacuum tube and frozen. The study protocol was approved by the Institutional Review Board of Indiana University. Informed consent was obtained from the parents of all of the subjects and from the subjects themselves if they were 8 yr of age or older. Genomic DNA was extracted from blood using a QIAamp Blood Midi Kit (QIAGEN, Valencia, CA). LHX3 exons were amplified by the PCR using primers recognizing intronic sequences as we have described (Fig. 1B
) (24). Each PCR contained 2.5 U PfuUltra DNA polymerase (Invitrogen, La Jolla, CA), 10 mM dATP, dCTP, dGTP, and dTTP, approximately 200 ng genomic DNA, and 10 pmol of each forward and reverse primer. Primers to amplify each exon were as follows: exon Ia, 5'-tgacctcggaggagcgcgtct-3' and 5'-caaccgctgtcccgcactctt-3'; exon Ib, 5'-gaaagttcgggactggagagt-3' and 5'-cagtgccacaacctcactca-3'; exon II, 5'-tacgaggtgacccagaactt-3' and 5'-cctggccttggtgattgtga-3'; exon III, 5'-tttcagaccaggaaaggtgg-3' and 5'-cgaaatgagcctcgcgcttc-3'; exons IV and V, 5'-gctgccgcgcctcaccgct-3' and 5'-aggagtccactaactccatg-3'; and exon VI, 5'-cgctgactgagcctctgctt-3' and 5'-cctcgtgtgaggtgcagggt-3'. Touchdown PCR cycling parameters were as follows: 94 C for 2 min; 94 C for 10 sec, 66 C for 10 sec, and 72 C for 2 min for two cycles; 94 C for 10 sec, 64.5 C for 10 sec, and 72 C for 2 min for two cycles; 94 C for 10 sec, 63 C for 10 sec, and 72 C for 2 min for two cycles; 94 C for 10 sec, 61.5 C for 10 sec, and 72 C for 2 min for two cycles; and 94 C for 10 sec, 60 C for 10 sec, and 72 C for 2 min for 20 cycles. If required, reactions amplifying GC-rich regions were supplemented with MasterAmp PCR optimization buffers (Epicenter, Madison, WI). Reaction products were analyzed on 1.5% agarose, Tris-borate gels and subsequently ligated into the pCR4-TOPO-Blunt vector (Invitrogen, Carlsbad, CA). Plasmid minipreps were performed, and insert-containing plasmids were sequenced on both strands by automated DNA sequencing using a Perkin-Elmer DNA sequencer (Biochemistry Biotechnology Facility, Indiana University School of Medicine) and M13 forward and M13 reverse primers. At least six PCR products of each amplified exon were sequenced from each patient to ensure more than 98% confidence of characterization of both alleles. The candidate mutation in exon II of patient AM was then confirmed by sequencing of amplicons representing another two independent PCRs.
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Site-directed mutagenesis
The deletion mutation was introduced into expression vectors containing human LHX3a and LHX3b cDNAs (9) using the QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA) as we have described (26). Mutagenic oligonucleotide sequences were 5'-gagagatcccgctgtgcgcggctgtgaccagcacatcc-3' and 5'-ggatgtgctggtcacagccgcgcacagcgggatctctc-3'.
In vitro transcription/translation
Radiolabeled wild-type and mutant LHX3 proteins were synthesized in vitro from pcDNA3.1-based expression vectors by a modification of methods that we have described (22). Translation reactions included plasmid substrates, T7 RNA polymerase, TnT rabbit reticulocyte lysates (Promega, Madison, WI), and [35S]cysteine (GE/Amersham, Piscataway, NJ). Labeled proteins were separated using SDS-PAGE followed by treatment of gels with Amplify fluorography reagent (Amersham) and visualized using a Storm phosphorimager (GE/Amersham/Molecular Dynamics).
Patient description
The patient is a 6-yr, 9-month-old male. He was born full-term with a birth weight of 2.8 kg (5th percentile) and a length of 46.6 cm (<5th percentile) to healthy consanguineous parents of Sephardic-Jewish descent originally from Azerbaijan (Fig. 1
). He presented on the first day of life with cyanosis, poor feeding, persistent jaundice, and a micropenis (stretched penile length of 1.5 cm). His examination showed a large anterior and posterior fontenelle, mild pulmonic stenosis, and hyperteleorism. Initial evaluation was consistent with central hypothyroidism (Table 1), and thyroid replacement therapy was started at d 4 of life. The patient remained hospitalized for poor weight gain. Serum IGF-I and IGFBP-3 levels were low, consistent with GH deficiency. A random serum cortisol was normal at 2 wk of age (Table 1). During the first year of life, the patient lacked the physiological LH, FSH surge, suspicious for gonadotropin deficiency (Table 1). Cryptorchidism was noticed, and orchidopexy was performed at 1 yr of age. The peak of ACTH rise was normal after CRH stimulation, consistent with normal corticotroph function (Table 2).
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180°). He was diagnosed with mental retardation by school psychoeducational assessment and is currently functioning 30–40% behind an age-appropriate level, particularly in reading and math skills. He is unable to speak words clearly or to construct sentences. The speech delay is a result of the global mental retardation. He is receiving speech, physical, and occupational therapy.
On neurological exam, there was mild hypotonia and immature prehensile skills. There was a suggestion of bilateral deltoid weakness and weakness in neck muscles including sternocleidomastoids. Mild weakness was also noted at the pelvic girdles, but the gait was normal and no fasciculations were seen. The cranial nerves were normal and the tendon reflexes were preserved.
Electromyography (EMG) of median, ulnar, and common peroneal nerves and deltoid, biceps, and vastus lateralis muscles was performed. EMG of the deltoid muscle showed moderate fasciculation potentials and occasional fibrillation potentials suggestive of a dysfunction at the anterior horn cell. Motor units and recruitment could not be adequately assessed because of poor cooperation. The paraspinal EMG could not be assessed because of poor cooperation. Nerve conduction studies were normal.
The parents’ anterior pituitary hormone panel (PRL, IGF-I, free T4, TSH, LH and FSH) was normal. The father’s brother died in childhood, and the mother’s twin sister died during infancy of unknown causes.
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Results |
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TopAbstractIntroductionPatient and MethodsResultsDiscussionReferences |
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The hormone deficiency profile and neck abnormality of the patient suggested that it may be caused by an inactivating mutation of the LHX3 gene. To test this hypothesis, genomic DNA was extracted from a peripheral blood sample and all LHX3 gene exons were amplified by the PCR using primers recognizing intronic sequences (Fig. 1B). Sequence analysis of cloned PCR products revealed a deletion of a single thymidine residue in exon II of both alleles (Fig. 1C). The mutation is g.159delT based on GenBank reference sequence AF367086 (the mutation is referred to as exon II delT hereafter). All other exons were normal in patient IV-2, and the entire gene was normal in an unrelated patient with similar hormone deficiencies but without the limited neck rotation (Fig. 1C and data not shown). The mutation was then confirmed by sequencing of amplicons representing another two independent PCRs using genomic DNA from patient IV-2.
The genotypes of the parents of patient IV-2 and his two sisters were then determined. Three approaches were taken. First, exon II of LHX3 was amplified from genomic DNA samples and cloned amplicons were sequenced as described above. Second, exon II amplicons were directly sequenced in bulk to determine the genotype. Lastly, because the observed mutation disrupts a Cac8I restriction site in exon II, these amplicons were digested with this restriction endonuclease, and reactions products were separated by electrophoresis. These experiments demonstrated that the parents of patient IV-2 were heterozygous for the LHX3 exon II delT mutation, and his sisters (IV-1 and IV-3) were both homozygous for the wild-type allele (Fig. 1, A and D, and data not shown).
The LHX3 exon II delT mutation found in patient IV-2 is predicted to cause a frame shift in translation of proteins from LHX3 mRNAs causing the production of short, approximately 18-kDa protein products lacking the LIM domains and the homeodomain of LHX3 (Fig. 3A). To confirm that the deletion would have this effect, the mutation was engineered into expression vectors containing full-length human LHX3a and LHX3b cDNAs by site-directed mutagenesis. These plasmids were used as substrates for in vitro transcription/translation reactions using rabbit reticulocyte lysates and [35S]cysteine (as a source of radiolabel). Labeled proteins were separated by electrophoresis and visualized using a phosphorimager. As predicted, wild-type LHX3a migrated with an apparent mass of approximately 44 kDa, and the protein from the mutated cDNA migrated with an apparent mass of approximately 18 kDa (Fig. 3B). Similar data were observed for LHX3b (data not shown). In these experiments, the signal from the protein produced from the mutated cDNA is notably weaker than the wild-type protein (Fig. 3B). This result is likely a result of reduced labeling of the mutant protein because it has less than half the number of cysteines present in the wild-type proteins. It is also possible, however, that the aberrant protein has decreased stability.
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Discussion |
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). This patient despite neck rigidity also has a normal cervical spine configuration and normal alignment in contrast to at least one of the previously reported patients who displayed an abnormal steepness of the cervical spine (20). In the previous report, the EMG was normal in two patients (20); however, the EMG findings in our patient suggest an anterior horn cell disease process that may be localized to the lower cranial and cervical anterior horn cells, but there is otherwise no overt cranial nerve involvement. This assumption is based on the presence of moderate fasciculations; unfortunately, motor unit morphology could not be obtained because of poor patient cooperation. In summary, the patient has mental retardation and a webbed neck with the suggestion of weakness about the rigid neck muscles. The mutation may result in focal amyotrophy, and other patients with LHX3 mutations will need to be assessed with detailed EMG studies to determine whether this represents an associated neurological phenotype. In mice, Lhx3 is expressed in several regions of the nervous system including the brain stem, pons, medulla oblongata, pineal, and hindbrain (11, 12, 13). Thus, the association between LHX3 mutations and nervous system pathologies such as amyotrophies, speech difficulties, and mental retardation is plausible, but additional investigations in other subjects with LHX3 mutations are needed.
Two different mutations were identified in the first report; three patients from one family had an A to G transition that resulted in a Y111C nonconservative substitution in the second LIM domain of LHX3a (Y116C in LHX3b) (20). Another patient from an unrelated family had a homozygous deletion of 23 bp involving the last three bases of exon 3 and the adjacent splice-donor site (20). The inheritance pattern for both mutations was found to be autosomal recessive (20). The patient reported here is homozygous for g.159delT (based on reference sequence AF367086) in exon 2 of LHX3, having inherited a copy of the recessive mutated gene from his parents who are both heterozygous.
The LHX3 gene exon II delT mutation described here causes a frame shift that is predicted to result in an altered reading frame and the addition of 139 aberrant amino acid residues after the 32nd residue of LHX3a (Fig. 3A). The aberrant protein is ended by the occurrence of a premature termination codon that lies in exon 4 of the gene, resulting in a short protein product of approximately 18 kDa that is indeed observed after transcription/translation of LHX3 cDNAs into which the deletion has been engineered (Fig. 3B). This protein would lack the LIM domains and the DNA-binding homeodomain and would therefore be predicted to be functionally inactive, as we have previously described for a truncated form of LHX3 (22). In vivo, it is possible that no significant protein product is made because of nonsense-mediated decay of LHX3 mRNAs. The location of the premature termination codon in the fourth exon, i.e. in a location 5' to the last 50 nucleotides of the penultimate exon is consistent with the likely targeting of this condition for nonsense-mediated decay (29, 30). However, there are exceptions to the defined rules for nonsense-mediated decay, and some transcripts appear to be especially prone or resistant to this process (29, 30), so the outcome in this case is speculative. The normal phenotype of the heterozygous parents of the described patient is consistent with the idea that, if any protein is made, the produced peptides are inactive, rather than having any significant negative activity. In either case, the evidence favors the hypothesis that this patient lacks LHX3 gene function.
The effects of pituitary transcription factor gene mutations on pituitary cell growth, both prenatally and after birth, appear to be complex. In the original report of LHX3 gene mutations, although some patients were described with hypoplastic pituitaries, one patient developed an enlarged anterior pituitary gland later in life (20). By contrast, some patients with mutations in the PROP1 gene (a gene encoding Prophet of Pit-1, a key pituitary transcription factor) display hyperplastic pituitary glands that later become hypoplastic (31). Longitudinal studies of pre- and postnatal pituitary development in normal and Prop1-deficient dwarf mice have examined the biology underlying this observation (32). These experiments reveal that fetal pituitaries lacking Prop1 function have an abnormal morphology because mutant anterior pituitary progenitor cells fail to leave the perilumenal region of Rathke’s pouch and do not differentiate (32). Later, the lack of progenitors in the anterior lobes of the mutant pituitaries is associated with enhanced apoptosis and reduced proliferation (32). The LHX3-deficient patient described here appears to perhaps have abnormal proliferation of pituitary cells, and this finding was observed both shortly after birth and more than 6 yr later. LHX3 has been implicated in the proliferation of the hormone-secreting cell types during pituitary development in mice (15). It is possible, however, that under some circumstances, an absence of LHX3 function could also result in inappropriate proliferation of pituitary cells. Further study of the nature of the lesion present in this patient would be necessary to provide additional data supporting this idea.
This study demonstrates that there is phenotypic variation in CPHD patients with mutations in LHX3. The actions of other genes within the genomes of these patients are likely to affect the phenotypic outcome of inactivating LHX3 mutations. Studies in animal models have demonstrated the influence of genetic background on the phenotypes associated with an individual (mutant) pituitary transcription factor allele. For example, it has been reported that the pituitary disease phenotypes associated with a mouse Lhx4–/– genotype are variable (33). In addition, the phenotype of homozygous mice carrying a null allele of Prop1 is markedly influenced by the genetic background (34). Genetic modifiers therefore likely play a pivotal role in the disease symptoms of patients with inactivating LHX3 mutations.
This study confirms the importance of LHX3 gene function in pituitary development and is consistent with a role in the development of the human nervous system. More detailed clinical case reports with special attention to cognition and detailed EMG studies will elucidate the role of this mutation in neuronal development and function. The presence of a hypointense pituitary lesion, possible amyotrophy, and mental retardation in this patient extends the phenotypic profile associated with LHX3 gene mutations.
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Acknowledgments |
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Footnotes |
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Abbreviations: CPHD, Combined pituitary hormone deficiency; EMG, electromyography; IGFBP-3, IGF binding protein-3; MRI, magnetic resonance imaging; PRL, prolactin.
This work was supported by grants to S.J.R. from the National Institutes of Health (HD42024) and the National Science Foundation (IBN 0131702). J.J.S. was an Elizabeth Steele Creveling Memorial Scholar during this work.
A.P.S.B., S.P., S.T., C.S.H., J.J.S., and S.J.R. have nothing to declare. E.C.W. has equity interests in Genentech and has received lecture fees from Eli Lilly and Co. H.A. has received lecture fees from Animas, Genentech, Eli Lilly, and Pfizer and has received consultation fees and is on an advisory board of Animas, NCGS, Eli Lilly, and Serono.
Received October 27, 2005.
Accepted December 27, 2005.
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References |
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-subunit promoter. J Biol Chem 276:19020–19026This article has been cited by other articles:
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F. Castinetti, A. Saveanu, R. Reynaud, M. H. Quentien, A. Buffin, R. Brauner, N. Kaffel, F. Albarel, A. M. Guedj, M. El Kholy, et al. A Novel Dysfunctional LHX4 Mutation with High Phenotypical Variability in Patients with Hypopituitarism J. Clin. Endocrinol. Metab., July 1, 2008; 93(7): 2790 - 2799. [Abstract] [Full Text] [PDF]
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 D. Kelberman and M. T. Dattani Hypothalamic and pituitary development: novel insights into the aetiology Eur. J. Endocrinol., August 1, 2007; 157(suppl_1): S3 - S14. [Abstract] [Full Text] [PDF]
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R. W. Pfaeffle, J. J. Savage, C. S. Hunter, C. Palme, M. Ahlmann, P. Kumar, J. Bellone, E. Schoenau, E. Korsch, J. H. Bramswig, et al. Four Novel Mutations of the LHX3 Gene Cause Combined Pituitary Hormone Deficiencies with or without Limited Neck Rotation J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1909 - 1919. [Abstract] [Full Text] [PDF]
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