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Arquivos Brasileiros de Endocrinologia & Metabologia

On-line version ISSN 1677-9487

Arq Bras Endocrinol Metab vol.52 no.8 São Paulo Nov. 2008

https://doi.org/10.1590/S0004-27302008000800004 

CLINICAL CASE REPORT

 

Absence of mutations in Pax6 gene in three cases of Morning Glory syndrome associated with isolated growth hormone deficiency

 

A ausência de mutações no gene PAX6 em três casos de síndrome de Morning Glory associada à deficiência isolada de hormônio do crescimento

 

 

Gil Guerra-JuniorI; Angela Maria Spinola-CastroII; Adriana A. Siviero-MiachonII; Roberto Gomes NogueiraIII; Sofia Helena V. Lemos-MariniI; Lilia Freire Rodrigues D'Souza-LiI; Priscila Cristina da SilvaIV; Emerson Salvador S. FrançaIV; Fernanda Caroline SoardiIV; Maricilda Palandi de MelloIV

IDepartment of Pediatrics, Pediatric Endocrinology Unit, Faculty of Medical Sciences, University of Campinas (Unicamp), Campinas, SP, Brazil
IIDepartment of Pediatrics, Pediatric Endocrinology Unity, School of Medicine, Federal University of Sao Paulo (Unifesp/EPM), Sao Paulo, SP, Brazil
IIIDepartment of Radiology, School of Medicine, Unifesp/EPM, Sao Paulo, SP, Brazil
IVCenter of Molecular Biology and Genetic Engineering (CBMEG), Unicamp, Campinas, SP, Brazil

Correspondence to

 

 


ABSTRACT

Morning glory syndrome (MGS) is a congenital optic disc dysplasia often associated with craniofacial anomalies, especially basal encephalocele and hypopituitarism. Clinical signs are varied and often occult. The PAX6 gene is involved in ocular morphogenesis and is expressed in numerous ocular tissues during development especially in the developing central nervous system. The aim of the present study is to evaluate PAX6 in MGS associated with isolated growth hormone deficiency. Three pre-pubertal males (A, B and C) with MGS and short stature due to growth hormone deficiency, treated with recombinant human growth hormone with limited response, were reported. Two of them had basal encephalocele. Coding and non-coding sequences corresponding of PAX6 different transcripts were analyzed by direct sequencing. Nucleotide variations causing putative aminoacid change were not observed. Patient A presented the new IVS2+9G>A transition, whereas patients A and C were heterozygous for known single nucleotide polymorphisms (SNP) within the intron 4. In addition, two SNP heterozygoses were observed for patient C in both intron 9 and 13. Sequencing also revealed several nucleotide variations in patient B. Two heterozygoses for known polymorphisms were identified along with a novel C>A nucleotide change in intron 4. This patient also presented a low number on the TG repeat in intron 9 and a new IVS11+33A>T transversion. Gene regulation and transcription of PAX6 are complex processes; there are two major protein isoforms, PAX6(-5a) and PAX6(+5a), and nine transcripts described. Furthermore, extra transcription regulatory elements have been postulated within PAX6 introns. Considering that neither population distributions on PAX6 polymorphism nor their linkeages with diseases have been reported, a functional effect due to alterations described here cannot be discarded.

Keywords: Encephalocele; Growth hormone; Morning glory syndrome; PAX6 gene; Short stature


RESUMO

A síndrome de Morning Glory (SMG) é uma displasia óptica congênita associada a anomalias craniofaciais, principalmente encefalocele basal e hipopituitarismo. Os sinais clínicos são variados e muitas vezes ocultos. O gene PAX6 está envolvido na morfogênese ocular e se expressa em vários tecidos oculares durante o desenvolvimento do sistema nervoso central. O objetivo deste estudo foi avaliar o gene PAX6 na SMG associada com deficiência isolada de hormônio de crescimento. Foram relatados três pacientes pré-púberes (A, B e C) com SMG e baixa estatura por deficiência de hormônio de crescimento tratados com hormônio de crescimento recombinante humano. As seqüências codificadoras e não-codificadoras correspondentes ao PAX6 em diferentes transcritos foram analisadas por seqüenciamento direto. Variações nucleotídeas com trocas pontuais de aminoácidos não foram encontradas. O paciente A apresentou uma transição nova IVS2+9G>A, enquanto os pacientes A e C foram heterozigotos para um polimorfismo (SNP) no íntron 4. Ainda, dois SNPs em heterozigose nos íntrons 9 e 13 foram observados no paciente C. O seqüenciamento também mostrou várias variações nucleotídeas no paciente B. Dois SNPs conhecidos com a alteração nucleotídea nova C>A no íntron 4 foram observados em heterozigose. Este paciente também apresentou um baixo número de repetições TG no íntron 9 e uma nova transversão IVS11+33A>T. A regulação e a transcrição do gene PAX6 são um processo complexo; existem 2 isoformas principais da proteína, PAX6(-5a) e PAX6(+5a) e 9 transcritos descritos. Considerando que nem a distribuição de SNPs no PAX6 e nem as suas ligações com as doenças foram relatadas, um defeito funcional devido às alterações descritas não pode ser descartado.

Descritores: Encefalocele; Hormônio do crescimento; Morning glory syndrome; Gene PAX6; Estatura baixa


 

 

INTRODUCTION

Morning glory syndrome (MGS) is a congenital malformation of the optic disc characterized by an enlarged, funnel-shaped optic disc and an elevated pigmented peripapillary tissue annulus, firstly described in 1970 and named by Kindler (1) due to a resemblance to the flower of the same name. MGS can be associated with basal encephalocele and hypopituitarism. Basal (meningo) encephalocele is an sporadic and occult malformation, described in 67,7% of MGS patients, usually associated with midline defects, such as cleft palate and corpus callosum agenesis, optic disc and other brain anomalies, and hormonal disturbance (2-5).

Azuma and cols. (6) identified PAX6 (paired box 6) gene (11p13) mutations in pedigrees with optic nerve malformations, including coloboma, morning glory disc anomaly, optic nerve hypoplasia/aplasia and persistent hyperplastic primary vitreous. Some of these mutations may affect transcriptional repression of the PAX2 promoter mediated by PAX6. In view of the detection of PAX2 gene mutations in papillorenal syndrome, it has been suggested that modifications in PAX2 function, as a result of PAX6 mutations, may affect some phenotypic expressions including those of the optic nerve malformations. Furthermore, Marquardt and cols. (7) demonstrated that PAX6 controls the transcriptional activation of retinogenic basic helix-loop-helix factors that predisposes subsets of multipotent retinal progenitor cells (RPCs) towards different retinal cell fates, thereby mediating the full retinogenic potential of RPCs. Nonetheless, the molecular mechanisms mediating the RPCs retinogenic potential have not been established yet.

Kioussi and cols. (8) showed that PAX6 is also implicated in the development of both, Rathke pouch and early anterior pituitary gland, whereas its expression controls the boundaries of somatotrope, lactotrope, and thyrotrope cell types. In the absence of PAX6 the thyrotrope cells lineage increases markedly along with the decrease of somatotrope and lactotrope cells.

The aim of the present study is to evaluate molecular features in PAX6 gene in three patients with MGS and isolated growth hormone deficiency.

 

SUBJECTS AND METHODS

Case reports

Three patients with MGS, two from Federal University of Sao Paulo (Unifesp/EPM – Brazil) (A and B) and one from University of Campinas (Unicamp – Brazil) (C) were referred to the Pediatric Endocrine Clinic, due to short stature. The ophthalmological exam, including a retinography, showed typical abnormalities of the MGS, comprising an optical disk with an enlarged choanoid and cupped aspect, with a pink pigmentation and a central white mass which hid the way of the vessels at the bottom of the disk. The disk was surrounded by a little elevated grey ring, with irregular borders and mixed with colored areas. The vessels were multiple, thin and radiated (Figure 1).

 

 

All patients were normally delivered at full term, after uneventful pregnancies. Patients A and B had unremarkable medical histories, but patient C presented failure to thrive and delayed psychomotor development, beginning at one year of age. None of them had family history of hormonal disorders or congenital anomalies. Clinical and laboratory data are summarized in Table 1.

 

 

During follow-up, all patients showed low growth rate (below 3 cm/year) and a hormonal profile compatible with isolated growth hormone deficiency (Table 1). They had no symptoms of diabetes insipidus. A magnetic resonance imaging (MRI - Phillips Gyroscan 1,5T), with sagittal and coronal images from the sellar region, was obtained on the T1 weighted spin echo (T1WSE) sequence before and after the paramagnetic contrast medium intravenous administered, and in T2 weighted spin echo sequence. It was observed inferior and posterior herniation of the sella content through the sella floor defect towards the sphenoidal sinus (basal encephalocele) in patients A and B. Patient C did not present encephaloceles, but he had a hypoplastic adenohypophysis, ectopic neurohypophysis with a normal stalk (Figure 2).

 

 

Patient A started growth hormone replacement therapy (rhGH) at age of 10, at a dose of 0.03 mg/kg/day, by subcutaneous injections, presenting with a growth rate of 11.5 cm/year during the first year of treatment and a progressive decrease, onward to 5 cm and 3 cm, respectively at the 2nd and 3rd year of treatment, regardless of an increase in the rhGH dosage. Patient B started rhGH at the age of 13 at a dose of 0.03 mg/kg/day. During the first two years of therapy the growth rate was respectively 12 cm/year and 5.0 cm/year, even though rhGH dosage was increased up to 0.05 mg/kg/day at the second year. Patient C was started on rhGH at the age of nine, at a dose of 0.05 mg/kg/day. During the first two years of treatment, growth rate was respectively 6.5 cm/ year during the first year and subsequently 5 cm/year. It is worth noting that during the second year the dosage of growth hormone was increased to 0.075 mg/kg/day. No other hormonal deficiencies were observed. Patient C showed a progressive ocular dysfunction and became blind at the right eye. The replacement therapy was discontinued after the third year. The presence of anti-GH antibodies was ruled out in the three cases. Currently, patients A and C are almost near final height and end up very short (height scores below -2 SDS). Patient B is still under rhGH therapy despite poor response.

Molecular studies

Genomic DNA was isolated from blood leukocytes using standard techniques (9). All fourteen coding and non-coding exons of the human PAX6 gene were amplified by PCR using specific primers listed in Table 2. PCR reactions were carried in 50 µl containing: ~500 ng genomic DNA, 10% DMSO, 200 µM each dNTP, 20 pmoles of each primer, 1xTaq buffer solution (Invitrogen, CA. USA) and 1.5 units of recombinant Taq DNA polymerase (Invitrogen, CA. USA). In general, cycling profiles were: 94 ºC for 5 min; 94 ºC for 1 min, Ta (ºC) specific for each primer pair (Table 2) for 1 min, 72 ºC for 1 min (30 cycles); and a final extension step of 72 C for 5 min. PCR products were purified with Wizard SV Gel and PCR Clean-UP System (Promega, Madison, WI, USA) and directly sequenced in both sense and antisense orientations using amplification primers; for long fragments, additional sequencing with an internal primer was performed (Table 2). Sequencing reactions were conducted using Big DyeTM Terminator Cycle Sequencing Kit V3.1 Ready Reaction (ABI PRISM / PE Biosystems, Foster City, CA, USA) and DNA sequences were obtained in an ABI PRISM® 3700 DNA Analyzer (ABI PRISM/PE Biosystems, Foster City, CA, USA). The electropherograms were analyzed by Chromas Lite program (free-software). Sequencing data obtained in this study was compared to the PAX6 gene sequence described by International Human Genome Consortium (GENEBANK access # NC_000011) using the GeneRunner v3.05 (free-software).

 

 

RESULTS

Genomic sequences corresponding to PAX6 different transcripts were analyzed by direct sequencing. PAX6 gene is transcribed into nine different transcripts. They differ to each other by alternative splicings of exons 1-4 in the 5'-UTR. Whereas sequencing of PAX6 coding DNA did not reveal any putative aminoacid change, new variations and several already known SNPs were found in non-coding regions (Table 3).

Patient A was found to be heterozygous for a novel IVS2+9G>A transition in intron 2. Two novel polymorphisms were observed for patient B: transversions g.14807C>A and IVS11+33A>T located in intron 4 and 11, respectively. Conversely, patient C did not present any novel nucleotide variation.

Patients A and C showed heterozygosis for the g.14592C>A (SNP-rs694617) transversion and also for the indel g.15449_15450insG (SNP-rs5790867) both located in intron 4. In addition, patient C presented the g.24214A>T (SNP-rs2239789) and IVS13+43T>G (SNP-rs3026393) transversions in intron 9 and 13, respectively.

Several SNPs were also observed for patient B. The heterozygosis for the nucleotide changes g.14806T>G (SNP-rs3026368) and g.15003A>G (SNP-rs3026369) within intron 4 were found, additionally only this patient was homozygous for +G allele at the indel g.15449_15450insG (SNP-rs5790867).

An intron 9 microsatellite formed by TG Short Tandem Repeat (STR) was also investigated. Normally, PAX6 alleles bear a sequence of (TG)19-29 repeats interrupted by a GG dinucleotide and followed by a (TG)7 repeat. Patients A and C were heterozygous for (TG)19/21 – GG – (TG)7 sequences, however patient B was heterozygous for (TG)16/19 repeats with absence of the intercalated GG dinucleotide and the last (TG)7 repeats (Table 3).

 

DISCUSSION

We reported three patients with ocular lesion compatible with MGS associated with isolated growth hormone deficiency with a limited response to rhGH replacement and PAX6 gene molecular features.

Neither hereditary nor pregnancy possible events according to published data so far (10) had influenced MGS in the cases described here. Reports on sex ratio have been controversial, but some authors consider MGS to be more commom in females (10,11). Nevertheless all patients studied in the present paper were males.

Likewise, the three described patients had bilateral disease whereas MGS is frequently unilateral and non-progressive. In addition, all patients became blind during the follow-up, which is a rare event (10-13). It was impossible to determine if rhGH have had any influence on the worsening the eye disease, since there are no reports available on this subject.

The patients in this report had mild midline defects such as hypertelorism (patient C), strabismus (patients A and C), and microphthalmia (patient B), however no signs of systemic diseases or any hormonal defect other than growth hormone deficiency was remarked. Most cases of MGS are considered as isolated ocular abnormalities, but other malformations of the skull, facial, mainly midline defects, and central nervous system may occur (13). Associations with genetic syndromes and systemic diseases, including growth hormone deficiency have also been reported (10,11).

The hormonal deficiency described in the other reported cases is usually secondary to the compression of the pituitary gland by basal encephaloceles (14,15), but the natural course of the hypothalamic-pituitary dysfunction is at present still unclear. In our patients the causative factors were not clearly identified. There is always a possibility that the growth hormone deficiency could have occurred by chance. Nonetheless, the growth hormone deficiency was not an isolated fact, and the patients did not have a good response to rhGH treatment, according to Ranke predictive models (16). There are no reports about other patients with MGS that had been treated with rhGH. Our patients had an acceptable response at the beginning of the treatment but a normal growth rate could not be maintained, even with a higher dose than usual, during the subsequent years. GH, IGF-1 and IGFBP-3 profile did not point out a possible GH resistance.

Regarding pituitary function, there was no progression of the hormonal deficiencies, even in what concerns diabetes insipidus, considered a common event associated with MGS (10,13).

In order to verify if mutations on PAX6 gene could explain MGS phenotypes in those patients, we proceed with PAX6 sequence analysis. However, nucleotide alterations were not identified in the protein coding sequence. PAX6 gene sequence analysis revealed several SNPs and at last one non-described heterozygous nucleotide was found in patients A and B. PAX6 protein is a transcription factor with important functions in the development of eyes and central nervous tissues (17). The most abundant transcripts present 13 or 14 exons depending on the inclusion or not of exon 5a. Therefore, there are two major PAX6 isoforms: PAX6(-5a), with 422 amino acids, and PAX6(+5a), with 436 amino acids resulting from the insertion of a 14 amino acid-long sequence in the paired domain (18). Furthermore, alternative splicings are responsible for the production of nine different transcripts, which results from distinct exon combinations in 5'UTR and are transcribed in different tissues, although most of them are mainly found in the cerebral tissue. Evidences suggest that the complexity of PAX6 transcription, regulation and expression is probably important for its multiple functions, whereas the spatiotemporal regulation and the expression of PAX6 isoforms are still not well understood. Recently, some elements corresponding to transcription regulatory consensus sequences have been described within PAX6 introns, suggesting that intronic sequences are important for the regulation maintenance in a high conserved gene (19). Concerning to SNPs already described, there is only one paper that failed to demostrate the linkage of SNP rs2239789 to myopia (20). In addition to the SNPs identified in the present study and those already described, PAX6 gene has a TG microsatellite within intron 9. Dinucleotide repeats located in introns are not generally considered to have functional roles in gene expression. However, their highly variable nature suggests a role as modifiers of RNA splicing. To support this idea, there is the finding that a variable number of TG repeats present in intron 8 of CFTR gene contributes to phenotype diversity in Cystic Fibrosis by forming RNA secondary structures that alter exon 9 splicing process (21). Linkages of TG repeats within intronic regions of different genes, including PAX7 which belongs to the same PAX6 gene family, have been described in a few diseases (22-24). Therefore, it is possible that nucleotide alterations within introns might have effects in transcript productions. Taking this hypothesis, PAX6 gene alterations found in patients A, B and C, whereas they cannot be considered as mutations, they could be probably associated with the phenotype. Considering that population studies on the distribution of PAX6 gene SNPs are not yet available in the database, a well conducted evaluation on normal individuals using novel and described SNPs must be performed to test this hypothesis. In summary, a functional effect of novel and known alterations described here for MGS patients cannot promptly be rejected.

Although MGS is usually a non-progressive untreatable disease, accurate diagnosis is important due to its association with systemic anomalies and increased incidence of retinal detachment (13). Undoubtedly, the disease manifests with a broad spectrum of characteristics, and we might have faced a new expression of the disorder. Notwithstanding, dysplasic optic disc should always be an alert for physicians to the occurrence of an encephalocele (25). Emphasis on these systemic and ocular associations may prompt early detection, and result in benefit from treatment, as well as could possibly be a clue for a better understanding of the disease under the molecular genetics aspects in order to develop a model for further studies.

No potential conflict of interest relevant to this article was reported.

 

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Correspondence to:
Gil Guerra-Junior
Faculty of Medical Sciences
PO Box 6111, Unicamp
13083-970 Campinas, SP, Brazil
E-mail: gilguer@fcm.unicamp.br

Received in 24/8/2008
Accepted in 14/10/2008

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