Pituitary stalk interruption syndrome in 83 patients: novel HESX1 mutation and severe hormonal prognosis in malformative forms

in European Journal of Endocrinology

Background

Pituitary stalk interruption syndrome (PSIS) is a particular entity in the population of patients with hypopituitarism. Only rare cases have a known genetic cause.

Objectives

i) To compare subgroups with or without extra-pituitary malformations (EPM) in a cohort of PSIS patients to identify predictive factors of evolution, ii) to determine the incidence of mutations of the known pituitary transcription factor genes in PSIS.

Study design

We analyzed features of 83 PSIS patients from 80 pedigrees and screened HESX1, LHX4, OTX2, and SOX3 genes.

Results

PSIS had a male predominance and was rarely familial (5%). Pituitary hypoplasia was observed only in the group with EPM. Multiple hormone deficits were observed significantly more often with versus without EPM (87.5 vs 69.5% respectively). Posterior pituitary location along the stalk was a significant protective factor regarding severity of hormonal phenotype. A novel HESX1 causative mutation was found in a consanguineous family, and two LHX4 mutations were present in familial PSIS.

Conclusion

PSIS patients with EPM had a more severe hormonal disorder and pituitary imaging status, suggesting an antenatal origin. HESX1 or LHX4 mutations accounted for <5% of cases and were found in consanguineous or familial cases.

Abstract

Background

Pituitary stalk interruption syndrome (PSIS) is a particular entity in the population of patients with hypopituitarism. Only rare cases have a known genetic cause.

Objectives

i) To compare subgroups with or without extra-pituitary malformations (EPM) in a cohort of PSIS patients to identify predictive factors of evolution, ii) to determine the incidence of mutations of the known pituitary transcription factor genes in PSIS.

Study design

We analyzed features of 83 PSIS patients from 80 pedigrees and screened HESX1, LHX4, OTX2, and SOX3 genes.

Results

PSIS had a male predominance and was rarely familial (5%). Pituitary hypoplasia was observed only in the group with EPM. Multiple hormone deficits were observed significantly more often with versus without EPM (87.5 vs 69.5% respectively). Posterior pituitary location along the stalk was a significant protective factor regarding severity of hormonal phenotype. A novel HESX1 causative mutation was found in a consanguineous family, and two LHX4 mutations were present in familial PSIS.

Conclusion

PSIS patients with EPM had a more severe hormonal disorder and pituitary imaging status, suggesting an antenatal origin. HESX1 or LHX4 mutations accounted for <5% of cases and were found in consanguineous or familial cases.

Keywords:

Introduction

Pituitary stalk interruption syndrome (PSIS) is characterized by the presence of a thin or absent pituitary stalk. It is commonly associated with a hypoplastic or aplastic anterior pituitary or an ectopic posterior pituitary. PSIS was identified by magnetic resonance imaging (MRI), which provides precise visualization of abnormalities in the hypothalamic and pituitary regions (1). This anatomical abnormality can be associated with other midline abnormalities and variable endocrine disorders, ranging from isolated GH deficiency (IGHD) to combined pituitary hormone deficiency (CPHD). The endocrine outcome seems to be a progressive onset of hormone deficiencies leading to panhypopituitarism (2); posterior pituitary function is usually maintained (3).

The causes of PSIS are still unknown. Former theories implicated traumatic birth, considering the high rate of perinatal events in this population (4). More recent studies favor organogenesis defects that may be of genetic origin or due to environmental factors during pregnancy (2, 5, 6). Over the last decade, mutations of transcriptional factor genes involved in pituitary development have been reported in human congenital hypopituitarism (7). Genetic screening for GH-N (GH1), GHRHR, POU1F1, LHX3, or PROP1 in several cohorts of patients with PSIS failed to reveal any mutation (3, 8, 9, 10, 11, 12). Rare mutations of HESX1, LHX4, and more recently OTX2 or SOX3 genes have been reported in PSIS (see review (7)). HESX1 mutations are responsible for a wide spectrum of clinical features, from IGHD with no midline forebrain or optic defect to midline brain defects as part of septo-optic dysplasia (SOD) (13, 14, 15, 16). LHX4 mutations have been reported to cause CPHD with variable neuroradiological abnormalities: PSIS, sella turcica or pituitary hypoplasia, ectopic posterior lobe, and defect of the corpus callosum and hindbrain (17, 18, 19). OTX2 gene defects, first observed in association with eye malformations and later with variable pituitary disorders, were recently reported in three PSIS patients with no ocular abnormalities (20, 21). Both over- or under-dosage of SOX3 may be associated with X-linked hypopituitarism, PSIS, and mental retardation, but two cases of SOX3 abnormality have been reported without mental delay (22, 23). Genetic mutations therefore seemed to be associated with extra-pituitary malformations (EPM). In this study, we analyzed clinical, hormonal, and neuroradiological features and conducted molecular analyses of HESX1, LHX4, OTX2, and SOX3 in a cohort of PSIS patients to define prognostic factors of the endocrine outcome, and to determine the frequency of genetic origin of this syndrome. We found that patients with EPM (EPM+) more frequently harbored a severe hormonal and imaging status than those without EPM (EPM−), and we identified LHX4 and HESX1 mutations only in familial or consanguineous cases.

Subjects and methods

Patients

Eighty-three patients were recruited from both national and international centers between 1996 and 2007: 69 patients through the GENHYPOPIT network, a multicentric study in pediatric and adult endocrinology centers for the screening of pituitary genetic determinants of CPHD, and 14 additional patients for whom DNA was not available. All patients presented with PSIS were defined on the basis of MRI findings showing a thin or absent pituitary stalk and no normal posterior lobe hypersignal in the sella turcica, and all had at least one anterior pituitary hormone deficiency, including GHD as defined below. The clinical features were collected by the use of a specific clinical report form. Detailed phenotypic characterization of these 83 patients (58 males) is shown in Table 1. Genomic DNA was screened from 69 patients for LHX4, HESX1, and OTX2 genes, and in males for SOX3. Written informed consent from all patients or parents of minors was obtained for this study approved by our institutional ethics committee.

Table 1

Clinical, perinatal, hormonal, and magnetic resonance imaging (MRI) features in 83 patients. Comparison between the EPM+ group (with extra-pituitary malformations) and the EPM− group (with no extra-pituitary malformation).

FeaturesPSIS patients (n=83)EPM+ (n=24, 28.9%)EPM− (n=59, 71.1%)P (EPM+ vs EPM−)
Mean age at diagnosis (years) (n=70, 21/49)9.6±8.89.4±11.69.51±7.3NS
Perinatal events
 IUGR (n=65, 17/48)9.2%11.8%8.3%NS
 Neonatal distress (n=63, 16/47)20.6%18.8%21.3%NS
 Breech presentation (n=61, 15/46)18%13.3%19.6%NS
Hormonal status (n=83, 24/59)
 IGHD6%8.3%5.1%NS
 CPHD
 GHD+1 anterior pituitary hormone deficit19.3%4.2%25.4%0.01*
 GHD+2 anterior pituitary hormone deficits15.6%20.8%13.6%NS
 GHD+3 anterior pituitary hormone deficits43.4%45.9%42.4%NS
 GHD+4 anterior pituitary hormone deficitsa15.7%20.8%13.5%NS
MRI
 Anterior pituitary (n=79, 23/56)
 Not visible 2.5%8.7%0%0.01*
 Hypoplasia77.2%69.6%80.4%NS
 Normal20.3%21.7%19.6%NS
 Posterior pituitary lobe (n=81, 24/57)
 Median eminence45.7%45.8%45.6%NS
 Along pituitary stalk13.6%16.7% 12.3%NS
 Invisible7%12.5%6%NS
 Ectopic unspecified32.1%25%35.1%NS
 Pituitary stalk (n=69, 21/48)
 Thin11.6%9.5%12.5%NS
 Interrupted40.6%47.6%37.5%NS
 Invisible47.8%42.9%50%NS
The P values for the EPM+ and EPM− groups by univariate comparisons were obtained by one-tailed Student's unpaired t-test (or Wilcoxon test if necessary) or when appropriate by one-tailed Pearson's χ2 test (or Fischer exact test if necessary). The values were considered significant when *P<0.05. Numbers in parentheses represent documented cases for each feature (n=total, EPM+/EPM−). SGA, small for gestational age; IGHD, isolated GH deficiency; CPHD, combined pituitary hormone deficiencies; ND, not documented; NS, not significant.

PRL status known for 76 patients (10 PRL deficits, 52 normal, 14 hyperprolactinemia).

The two groups were determined according to the presence (EPM+ group: 24 patients) or absence (EPM− group: 59 patients) of EPM, whether CN, ocular, dental, craniofacial, and/or cardiac. Cases with a family history of IGHD or CPHD with at least one PSIS case among first-degree relatives were considered as familial cases.

Clinical studies

Neonatal features and description of physical malformations were provided by the referring medical centers and interpreted according to gestational age, delivery conditions, and presence or absence of neonatal distress. Birth weight and length were expressed as SDS for gestational age and sex, and patients were defined as small for gestation age (SGA) when birth weight and/or length was below −2 SDS (8). Breech presentation and neonatal hypoxemia (defined by an Apgar score <7 at 5 min after birth or need for neonatal resuscitation) were recorded. Height at diagnosis was reported by each referring center and expressed in SDS according to Sempé et al. (24).

Hormonal studies were performed in all index patients, in each referring medical center. Normal values for each center were taken into account. The plasma GH response was studied with at least two provocative tests in each patient: insulin intolerance test (0.05 U/kg), GHRH infusion test (80 μg, Somatoreline; Choay/Sanofi, Gentilly, France), or propranolol–glucagon test (0.25 mg/kg propranolol orally and 1 mg glucagon, i.m.). A diagnosis of TSH deficiency (TSHD) was made if serum thyroxine (T4) concentration was subnormal (free T4 <12.0 pmol/l or total T4 <65 nmol/l) with an inappropriately low serum TSH concentration (<5 μU/ml). Basal plasma ACTH and cortisol were measured at 0800 h (normal range for cortisol: 210–560 nmol/l). The plasma ACTH and cortisol response were also determined during an insulin tolerance test. Gonadotroph axis was investigated only in patients of postpubertal age, i.e. over 15 years for female and 17 years in male subjects. FSH–LH deficiency was diagnosed on the basis of delayed or absent pubertal development with low serum testosterone or estradiol levels and blunted LH/FSH response to a GNRH stimulation test. Prolactin (PRL) deficiency and hyperprolactinemia were defined as basal plasma PRL levels lower than 5 ng/ml and above 25 ng/ml respectively. CPHD was defined as GHD associated with at least one other anterior pituitary hormone deficiency.

Pituitary MRI with gadolinium injection was performed in all patients, in each referring medical center according to standardized procedures, using precontrast sagittal and coronal spin echo T1-weighted images followed by post-gadolinium T1-weighted imaging (25). Pituitary height was measured and compared with normal value for age (25). The pituitary stalk was considered ‘thin’ when it had a continuous but extremely thin appearance with below normal size (1), or ‘interrupted’ when discontinuous, or ‘invisible’ when not observed on any section. The location of the ectopic posterior pituitary lobe was described either as localized at the median eminence, along the pituitary stalk, or not visible. Associated abnormalities of the brain, cerebellum, optic nerves, optic chiasm, sella turcica, or midline structures were also systematically sought. MRI films were not centrally read.

Molecular analysis of transcription factor genes

Genomic analysis of HESX1, LHX4, OTX2, and SOX3 was performed by direct sequencing. Genomic DNA was extracted from peripheral blood lymphocytes. The coding regions and intron–exon boundaries of HESX1, LHX4, and OTX2 as well as the single exon of SOX3 were amplified by PCR (Kit HotStarTaq DNA polymerase, Qiagen) using exon-flanking primers (Supplementary Table 1, see section on supplementary data given at the end of this article). The same primers were used for sequencing, with analysis on an AB3130xl (Applied Biosystem, Foster City, CA, USA).

Statistical analyses

Results are expressed as mean±s.d. for age and bone age or in percentages. Univariate comparisons between groups were performed by one-tailed Student's unpaired t-test (or Wilcoxon test if necessary) or when appropriate by one-tailed Pearson's χ2 test (or Fischer exact test if necessary). Multivariate studies were also performed as logistic regression (with descendant stepwise analysis based on likelihood ratio). P values were considered significant when <0.05.

Results

General features of PSIS patients

Eighty-three patients were investigated from clinical centers based in seven countries (France, n=67; Tunisia, n=8; Turkey, n=3; Algeria, n=2; Argentina, n=1; Egypt, n=1; and Lebanon, n=1) (Table 1). Sex ratio was 2.3 (58 males/25 females). Nine index cases (11.25%) were born from consanguineous parents, and four index cases (5%) had a familial history of CPHD.

Mean age at diagnosis was 9.6±8.8 years (1 day–39 years), with a height retardation of −3.5±1 SDS. Eighteen patients were diagnosed before or at 2 years of age, and five patients were diagnosed at adult age (>18 years). The mean bone age at diagnosis was 5.2±3.6 years, with a bone age retardation of −3.2±2.3 years. Patients were referred for growth retardation (86.8%), hypoglycemia (15.8%), jaundice (5.3%), and/or salt wasting (1.3%). Mean age at the study analysis was 21.8±11.6 years. All of the PSIS patients had GHD, 79.5% had deficiency in TSH, 67.5% in ACTH, 65.1% in LH/FSH, 14.5% in PRL, and 16.9% had hyperprolactinemia. IGHD was found in only 6% of patients. Among the 58 males, 23 presented micropenis (39.7%), and six had cryptorchidism (10.3%), presumably related to GH and/or LH/FSH deficiency.

Comparison of phenotype features in patients with (EPM+) or without (EPM−) EPM

Among these 83 patients, 24 (28.9%) referred to as EPM+ presented EPM (CN, ocular, dental, craniofacial, sella turcica, or cardiac malformations; Table 2); 59 (71.1%) had no EPM (EPM−). Mean age at diagnosis was similar for the two groups (EPM+: 9.4±11.6 years; EPM−: 9.6±7.2 years; P=0.46). The incidence of neonatal distress and breech delivery was not significantly different (18.8% EPM+ versus 21.3% EPM−, and 13.3% EPM+ versus 19.6% EPM− respectively) as shown in Table 1.

Table 2

Features of the EPM+ group, presenting extra-pituitary malformations.

No.SexPerinatal eventsHormonal statusAnterior pituitaryPosterior pituitaryPituitary stalkAssociated malformations
1FNDGHD+3 defectsHypoplasiaAlong PSThinAbsence of interventricular septum
2MNDGHD+3 defectsHypoplasiaEctopicNDHydrocephalia, macrosomia, blindness, SOD
3M0GHD+1 defect (PRL not done)NormalEctopicNDChiari 1 malformation
4M0GHD+3 defectsNormalMEInterruptedChiari 1 and chiasma malformations
5FBreechIGHDHypoplasiaMEInvisibleLeft optic nerve atrophy, amblyopy, SOD
6M0GHD+2 defectsHypoplasiaInvisibleInvisibleBilateral optic nerve atrophy, SOD
7FNDGHD+4 defectsHypoplasiaMEInvisibleCraniostenosis
8F0GHD+3 defectsHypoplasiaMEInterruptedEnlarged floor of the third ventricle
9F0GHD+3 defectsHypoplasiaInvisible InterruptedMyelinization defect
10MNDGHD+4 defectsHypoplasiaAlong PSInvisibleAmblyopy, nystagmus, SOD
11F0GHD+2 defectsHypoplasiaMEInvisibleHypoplasia of sella turcica
12MNDGHD+3 defectsNDEctopicNDOptic nerve hypoplasia, retinal cyst, coloboma, microcephalia
13MBreech NDGHD+3 defectsHypoplasiaEctopicInterruptedHydrocephalia, frontal atrophy, cervical rigidity, Chiari 1 malformation, Syringomyelia
14M0GHD+4 defectsNo pituitary tissue visibleMEInvisibleMidline abnormalities
15M0IGHDHypoplasiaAlong PSInterruptedChiari 1 malformation, hypoplasia of sella turcica
16M0GHD+3 defectsNormalEctopicInterruptedCerebellar atrophy, hypotonia, Little syndrome
17aMNDGHD+4 defectsHypoplasiaMEInvisibleHypoplasia of sella turcica, Chiari 1, persistent craniopharyngeal canal
18F0GHD+4 defectsHypoplasiaMEInvisibleLeft frontal porencephaly
19M0GHD+3 defectsHypoplasiaEctopicInterruptedCerebral anterior agenesy
20FSGAGHD+2 defectsNo pituitary tissue visibleMEInvisibleMicrocephalia, ventricle dilatation, microphthalmia, SOD corpus callosum, cerebellar and sella turcica abnormalities
21bMSGA. BreechGHD+4 defectsNormalMEInterruptedCardiac malformations: IVC, IAC
22MNDGHD+2 defects (PRL not done)HypoplasiaAlong PSInterruptedIncisor agenesy
23M0GHD+3 defectsNormalMEInterruptedCardiac malformations: IVC, Pulmonary stenosis
24aMNDGHD+2 defectsHypoplasiaInvisibleThinHypoplasia of sella turcica and of corpus callosum
ND, not documented. Sex: F, female; M, male; SGA, small for gestational age; IGHD, isolated GH deficiency; Along PS, along the pituitary stalk; ME, median eminence; SOD, septo-optic dysplasia; IVC, interventricular communication; IAC, interauricular communication; PRL, prolactin.

Patients with LHX4 mutations.

Patients with HESX1 heterozygous polymorphism.

Concerning hormonal status, the proportion of patients with IGHD was very low (6%) and similar in both groups (P=0.29). Univariate studies showed significantly more patients bearing GHD in association with at least two other hormonal abnormalities in the EPM+ group than in the EPM− group (87.5% EPM+ versus 69.5% EPM−, P=0.04) (Fig. 1). Multivariate studies confirmed these data, showing that EPM was an independent risk factor in terms of severity of hormonal status (odds ratio (OR)=6.4, 95% CI (1.13; 36.4); P=0.04), when adjusted on imaging covariate.

Figure 1
Figure 1

Hormone status in both groups of PSIS patients: EPM+, with extra-pituitary malformation (EPM) and EPM−, without EPM. The EPM+ group comprises 24 PSIS patients and the EPM− group comprises 59 patients. GH deficiency (GHD) +2 or more hormone abnormalities was significantly more common in the EPM+ group than in the EPM− group (87.5 vs 69.5%, P=0.04). IGHD, isolated GHD. *PRL status known for 76 patients.

Citation: European Journal of Endocrinology 164, 4; 10.1530/EJE-10-0892

On MRI, severe pituitary hypoplasia (lack of detectable residual pituitary tissue) was observed only in the EPM+ group (8.7%, P<0.05). Multivariate statistical studies showed that having a posterior pituitary along the pituitary stalk was a protective factor regarding severity of hormonal phenotype (OR=0.05, 95% CI (0.04; 0.602); P=0.02), when adjusted on the presence or absence of EPM.

Molecular analyses

Gene screening was performed in 69 PSIS patients from 65 pedigrees; among them, seven had a familial history of PSIS (four pedigrees) and nine were born from consanguineous parents (seven pedigrees). Among these 69 PSIS patients, we found one new HESX1 mutation. This HESX1 gene defect consists of a homozygous nonsense mutation leading to a stop codon in the second exon (c.325C>T, p.Arg109X) predicting a severely truncated protein (loss of 74 amino acids including the homeodomain). The propositus was a boy, born post-term with cesarean delivery from consanguineous parents of Turkish origin. At 1 year of age, he was referred to a pediatric department for recurrent hypoglycemia and growth retardation. He also had micropenis and cryptorchidism. He was treated for GH and ACTH deficiencies. At 14 years of age, endocrine deficits also included TSH, LH/FSH, and PRL deficiencies. Pituitary MRI was performed and showed a hypoplastic anterior pituitary gland, an ectopic posterior pituitary lobe located at median eminence, and an interrupted pituitary stalk (Fig. 2). No SOD, midline brain defects, or other malformations were observed. In the family of the propositus, we found the same mutation at heterozygous state in his father, his mother, one of his two sisters, and one cousin, who were all asymptomatic. Furthermore, in one patient (EPM+, patient 21, Table 2) with no familial or consanguinity history, a novel heterozygous HESX1 allelic variation was found: p. Ser67Thr (c.200G>C). Analysis of other members of the family showed that both the father and a sister, phenotypically normal, also had the same allelic variation, which was not carried by the mother and a brother. The new c.200G>C heterozygous HESX1 allelic variation affects a poorly conserved nucleotide. The serine to threonine substitution was suggested to have minor physico-chemical effects by in silico analysis with Alamut Software (Interactive-biosoftware, Rouen, France).

Figure 2
Figure 2

Pituitary MRI in a patient bearing an HESX1 gene mutation. MRI was performed at 25 years of age. Non-enhanced coronal (A and B) T1-weighted sequence depicting an interrupted pituitary stalk (arrow, panel A), hypoplastic anterior pituitary gland (above asterisk, panel A), and ectopic posterior pituitary (dotted arrow, panel B). (C) Coronal MRI scan of the pituitary of a normal child at the anterior pituitary and the pituitary stalk in the normal sella turcica.

Citation: European Journal of Endocrinology 164, 4; 10.1530/EJE-10-0892

One familial case (group EPM+, patient 17, Table 2) had an already reported (18) LHX4 gene defect: a heterozygous intronic point mutation involving the splice-acceptor site preceding exon5 (c.607-1G>C heterozygote). A second LHX4 heterozygous mutation (c.293_294insC, p.Thr99AsnfsX53) was recently reported by our group and confirmed as a functionally defective variant on the basis of in vitro studies (17): two brothers born from non-consanguineous parents presented somatolactotroph and thyrotroph deficiencies. MRI showed poorly developed sella turcica and pituitary hypoplasia for both, associated with a hypoplastic corpus callosum, thin pituitary stalk, and invisible posterior pituitary for the younger one (EPM+, patient 24, Table 2), but with eutopic posterior pituitary and normal corpus callosum for the older one. Their father carried the same heterozygous mutation. At age 28, he harbored a partial gonadotroph deficiency and a hyperplasic pituitary without pituitary stalk or posterior pituitary abnormalities on MRI (17).

We also identified two LHX4 variants (c.1108G>A heterozygote, p.Gly370Ser and c.296 C>T heterozygote, p.Thr90Met), with normal DNA-binding and transactivation properties in transfection studies as we reported (17). Moreover, 56.3% of patients presented the c.983G>A (p.Asn328Ser) LHX4 polymorphism in a heterozygous (40%) or homozygous (16.3%) way. This polymorphism represented 36.4% of all alleles studied.

No OTX2 mutation was identified, even in patients bearing eye defects. The OTX2 polymorphisms c.98-46C>A (rs2277499) and 3′UTR+10G>A (rs171978) represented 7.2 and 11.6% of all alleles studied respectively. No abnormality detectable by sequencing was found in SOX3 gene in the males from our series.

Discussion

In this study, we analyzed neonatal, hormonal, neuroimaging data, and genetic features from patients with PSIS to better characterize this syndrome of unknown pathogenesis. Several clinical points of interest appeared when comparing two groups, one without EPM and one with EPM (CN, ocular, dental, craniofacial, sella turcica, or cardiac malformations). First, the EPM+ group was associated with a more severe hormonal status, as found in other studies (3, 6). This calls for repeat, careful long-term follow-up in these patients. Second, the presence of EPM was unfortunately not associated with an earlier diagnosis in our subjects, although earlier diagnosis has been reported in previous studies (about 9.5 vs <6 years) (2, 3). Indeed, diagnosis in the overall population should have been made earlier, considering the proportions of neonatal events (20.6%), SGA (9.2%), hypoglycemia (16%), jaundice (5.3%), EPM (29.3%), micropenis (40.4%), and cryptorchidism (10.5%). Such neonatal events should lead to earlier evaluation of pituitary function and stalk morphology by MRI to preserve height and mental development (2). Third, the number of perinatal events did not differ between both groups, even though the EPM− group had a slightly higher proportion of neonatal distress and breech presentation. Fourth, the EPM+ group included a significantly greater proportion of patients with no visible anterior pituitary. No association was found between the location of posterior pituitary and presence of EPM, as described in other cohorts of PSIS patients (5, 6).

MRI pituitary aspect appears important for analysis and has been related to severity of hormonal status (3, 9, 26). Indeed, regardless of the presence or absence of EPM, a correlation has previously been reported between anterior pituitary function and anatomical characteristics of the hypothalamic–pituitary region, particularly a better functional prognostic value of the visibility of the pituitary stalk (5, 26, 27). In our population, posterior pituitary along the stalk represented a protective factor regarding severity of hormonal status. Note that, in the literature, location of ectopic posterior lobe was also associated with functional prognosis, with a greater number of hormone deficiencies when posterior lobe is localized at the median eminence or hypothalamic region (26).

In the last decade, PSIS has been described in patients with gene defects affecting pituitary transcription factors: HESX1,LHX4, OTX2, or SOX3 (7). The present study reports systematic genetic analysis in patients with PSIS on the basis of current knowledge on genes involved in this condition. First, we found a novel HESX1 homozygous nonsense mutation generating a truncated protein, resulting in total loss of homeodomain and co-repressor binding. This HESX1 p.Arg109X mutation leads to a severely truncated protein including the homeodomain that is involved in DNA binding and required to recruit components of N-CoR-associated co-repressors as reported by Dasen et al. (28). HESX1 homozygous mutations have most often been related to severe hormonal, imaging, and clinical presentation features, whereas heterozygous mutations are associated with a milder phenotype with the presence of different midline brain defects or optic nerve hypoplasia (29). Surprisingly, despite this severe homozygous genotype, the phenotype of our patient was relatively light, with no midline brain defect or SOD. To date in the literature, only one patient, also born from a consanguineous union, has been reported bearing HESX1 homozygous mutation with pituitary stalk abnormality but no midline brain defect or SOD (29). So HESX1 homozygous mutation may be associated with a less severe phenotype than previously considered. We also identified a novel heterozygous variation of HESX1 in a patient and in his unaffected father and sister. In silico analysis did not favor a functional impact of this allelic variation. We thus considered this monoallelic variation as unlikely to have a pathogenic significance. However, as this variation was not found among 100 allelic controls (although not population matched), we cannot rule out a variant of functional significance but incomplete penetrance.

We also found two LHX4 allelic variations of unknown significance (p.Gly370Ser and p.Thr90Met), and one common polymorphism of LHX4. Concerning the two variants of unknown significance, whereas Gly to Ser amino acid substitution was not predicted to have significant physico-chemical effects, Thr90Met amino acid substitution affects the LIM domain and is likely associated with important physico-chemical changes. In our in vitro studies, these variants did not modify the DNA-binding and transactivation properties of the protein (17). Considering the limitations of functional studies, however, it cannot be ruled out that these variants may somehow be functionally relevant. The overall incidence of LHX4 defects was very low (n=2, 2.9%), but it was high in a subset of patients with familial history of PSIS (50%). Including our results, 60% of cases with LHX4 mutation currently reported in the literature had familial history of CPHD (17, 18, 19). Note that the prevalence of familial CPHD with PSIS was similar in our cohort and in the cohort of Pinto et al. (3) (6.2 and 5.9% respectively) but lower than in the CPHD cohort without PSIS previously reported by our group (18 familial cases among 120 unrelated CPHD patients (15%)) (30).

The male preponderance observed in our study (sex ratio: 2.3/1) is in keeping with other studies (2, 3, 5, 9, 26) suggesting an X-linked inheritance. Neither HESX1, OTX2, nor LHX4 gene is located on X chromosome. Recently, X-linked inherited SOX3 abnormalities were reported to be responsible for hypopituitarism in patients with PSIS (22, 23). One genetic screening of SOX3 has been performed by Alatzoglou et al. (8) in 42 CPHD and 21 IGHD with ectopic posterior pituitary pedigrees without any sequence or gene copy number abnormalities. In our study, molecular genetics analysis through DNA sequencing revealed neither in-frame duplication nor deletion in the first polyalanine tract of the SOX3 gene, the only abnormalities responsible for molecular defects reported to date. Note that a large duplication cannot be excluded, as no cytogenetic study could be performed on patients' extracted genomic DNA collected through GENHYPOPIT.

Finally, no OTX2 mutation was found in our series. Heterozygous OTX2 mutations have been primarily reported in patients with highly variable ocular malformations and more recently hypopituitarism with or without ocular anomalies (20, 21). Dateki et al. (20) recently emphasized a low prevalence of OTX2 mutation in 61 Japanese patients with pituitary dysfunction without ocular anomalies, in line with our results.

Overall, the present study suggests that at least two clinical subsets of PSIS patients might be distinguished. The first one, with EPM, has more severe hormonal (CPHD) and pituitary imaging features, including pituitary hypoplasia. The second one, without EPM, has a higher proportion of neonatal distress but a lighter hormonal and pituitary imaging profile. This wide phenotypic spectrum might depend on genetic and/or environmental factors. Among this cohort of PSIS patients, we found only three unequivocally causative genetic defects. Noteworthy, mutation of HESX1 was found in a patient without SOD, born from one of the seven consanguineous unions reported in our study. Second, both mutations of LHX4 were found among the four index cases with a familial history of PSIS. Considering the rarity of familial or consanguineous cases of PSIS, HESX1 and LHX4 screening should thus be recommended in such a context. Our study shows that HESX1 or LHX4 gene defects can be ruled out as common causes of sporadic PSIS, a finding that does not currently support cost effectiveness of systematic genetic screening of these factors in this condition. We did not find any OTX2 mutation in this cohort, but only six patients harbored ocular malformations. OTX2 genetic screening should be performed when ocular malformation with or without hypopituitarism is present. However, pituitary phenotype in OTX2 mutations without ocular anomaly remains to be further studied.

Phenotype variability among PSIS patients and complexity of pituitary embryogenesis suggest that this syndrome involves several mechanisms, such as a multigenic pattern or, as in SOD, environmental influences (29). To date, the underlying mechanisms involved in most cases of PSIS thus remain to be identified. For PSIS, we thus recommend a careful and long-term follow-up with repeat hormonal evaluation, especially in patients with associated EPM, given the severity of hormonal prognosis in this group of patients. Molecular diagnostic analyses should be discussed according to familial or consanguinity status, and pattern of syndromic features.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/EJE-10-0892.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The present study was supported by a grant from Association pour le Développement des Recherches Biologiques et Médicales au Centre Hospitalier Régional de Marseille (ADEREM).

Acknowledgements

We thank the clinicians who sent blood samples of their PSIS patients for genetic screening in the GENHYPOPIT network: Dr M Abid (Sfax, Tunisia), Dr A Bennet (Toulouse, France), Dr P Berlier (Lyon, France), Prof. F Borzon-Chazot (Lyon, France), Prof. P Bouchard (Paris, France), Prof. P Bougneres (Paris, France), Prof. O Bruno (Buenos Aires, Argentina), Prof. P Caron (Toulouse, France), Prof. O Chabre (Grenoble, France), Prof. P Chatelain (Lyon, France), Prof. F Chentli (Algiers, Algeria), Prof. S Christin-Maitre (Paris, France), Prof. B Conte-Devolx (Marseille, France), Prof. M David (Lyon, France), Dr G Diene (Toulouse, France), Prof. M El Kholy (Cairo, Egypt), Dr O Evliyaoglu (Ankara, Turkey), Dr C Fedou (Montpellier, France), Dr A M Guedj (Nimes, France), Prof. G Halaby (Beirut Lebanon), Prof. M L Kottler (Caen, France), Prof. B Leheup (Nancy, France), Dr M Manavela (Buenos Aires, Argentina), Dr J C Mas (Nice, France), Prof. M Pugeat (Lyon, France), Prof. V Rohmer (Angers, France), Dr F Soumeya (Algiers, Algeria), Dr C Stuckens (Lille, France), Prof. A Tabarin (Bordeaux, France), Dr C Teinturier (Paris, France), Dr Z Turki (Tunis, Tunisia), and Dr M C Vantyghem (Lille, France). We also thank Anne Carle, Anne-Laure Germanetti, Morgane Pertuit, Nicole Peyrol, and Nadine Pluchino (Molecular Biology Laboratory) for the genetic analysis of transcription factors.

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*(R Reynaud and F Albarel contributed equally to this work)

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    Hormone status in both groups of PSIS patients: EPM+, with extra-pituitary malformation (EPM) and EPM−, without EPM. The EPM+ group comprises 24 PSIS patients and the EPM− group comprises 59 patients. GH deficiency (GHD) +2 or more hormone abnormalities was significantly more common in the EPM+ group than in the EPM− group (87.5 vs 69.5%, P=0.04). IGHD, isolated GHD. *PRL status known for 76 patients.

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    Pituitary MRI in a patient bearing an HESX1 gene mutation. MRI was performed at 25 years of age. Non-enhanced coronal (A and B) T1-weighted sequence depicting an interrupted pituitary stalk (arrow, panel A), hypoplastic anterior pituitary gland (above asterisk, panel A), and ectopic posterior pituitary (dotted arrow, panel B). (C) Coronal MRI scan of the pituitary of a normal child at the anterior pituitary and the pituitary stalk in the normal sella turcica.