HESX1 Mutations in Patients with Congenital Hypopituitarism: Variable Phenotypes with the Same Genotype

Clinical Endocrinology (2016) 85, 408–414 doi: 10.1111/cen.13067

ORIGINAL ARTICLE

HESX1 mutations in patients with congenital hypopituitarism: variable phenotypes with the same genotype

Qing Fang*, Anna Flavia Figueredo Benedetti†, Qianyi Ma*, Louise Gregory‡, Jun Z. Li*, Mehul Dattani‡, Abdollah Sadeghi-Nejad§, Ivo J.P. Arnhold†, Berenice Bilharinho Mendonca†, Sally A. Camper* and Luciani R. Carvalho†

*Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA, †Division of Endocrinology, Unit of Endocrinology and Development, Laboratory of Hormones and Molecular Genetics, Clinical Hospital of the Faculty of Medicine of the University of S~ao Paulo, S~ao Paulo, Brazil, ‡Developmental Endocrinology Research Group, Section of Genetics and Epigenetics in Health and Disease, Genetics and Genomic Medicine Programme, University College London, Institute of Child Health, London, UK and §Division of Pediatric Endocrinology, Floating Hospital for Children at Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA

Conclusion These three examples demonstrate that HESX1 Summary mutations cause variable clinical features in patients, which suggests an influence of modifier genes or environmental factors on Introduction Mutations in the transcription factor HESX1 can the phenotype. cause isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD) with or without septo- (Received 2 February 2016; returned for revision 22 February optic dysplasia (SOD). So far there is no clear genotype–pheno- 2016; finally revised 10 March 2016; accepted 16 March 2016) type correlation. Patients and Results We report four different recessive loss-of- function mutations in three unrelated families with CPHD and no Introduction midline defects or SOD. A homozygous p.R160C mutation was found by Sanger sequencing in two siblings from a consan- Congenital hypopituitarism refers to the deficiency of two or guineous family. These patients presented with ACTH, TSH and more pituitary hormones, and it is caused by mutations in one GH deficiencies, severe anterior pituitary hypoplasia (APH) or of several genes implicated in pituitary development, such as pituitary aplasia (PA) and normal posterior pituitary. The HESX1, OTX2, PROP1, POU1F1, LHX3, LHX4, SOX2, SOX3 1,2 p.R160C mutation was previously reported in a case with SOD, and GLI2. In some patients, the hormone deficiency may pre- CPHD and ectopic posterior pituitary (EPP). Using exome sent as part of a syndrome with abnormalities in structures that sequencing, a homozygous p.I26T mutation was found in a Brazil- share a common embryological origin with the pituitary gland, ian patient born to consanguineous parents. This patient had such as the eye and the forebrain. evolving CPHD, normal ACTH, APH and normal posterior pitu- HESX1 encodes a paired-like homeobox transcription factor 3,4 itary (NPP). A previously reported patient homozygous for p.I26T that was first identified in embryonic stem cells. HESX1 is one had evolving CPHD and EPP. Finally, we identified compound of the earliest known markers of the pituitary primordium. It heterozygous mutations in HESX1, p.[R159W];[R160H], in a can be detected in the anterior forebrain from 7Á5to8Á5 days patient with PA and CPHD. We showed that both of these muta- postcoitum (d.p.c.) and in the Rathke’s pouch from 8Á5to13Á5 tions abrogate the ability of HESX1 to repress PROP1-mediated d.p.c. Expression of HESX1 is important for the early determi- 5,6 transcriptional activation. A patient homozygous for p.R160H was nation and differentiation of pituitary gland, as well as normal 7,8 previously reported in a patient with CPHD, EPP, APH. forebrain formation in both mice and humans. A number of autosomal dominant and recessive mutations in HESX1 have been described in patients with a broad spectrum of Correspondence: Sally A. Camper, Department of Human Genetics, phenotypes ranging from isolated growth hormone deficiency University of Michigan Medical School, Ann Arbor, MI 48109-5618, USA. Tel.: 734-763-0682; Fax: 1-734-763-3784; E-mail: scamper@med. (IGHD), combined pituitary hormone deficiencies (CPHD) to 6,9–11 umich.edu septo-optic dysplasia (SOD). Magnetic resonance images and (MRI) also reveal variable effects. The anterior pituitary can be Luciani R. Carvalho, Endocrinology Discipline of Internal Medicine hypoplastic or aplastic, and the posterior pituitary can be ectopic Department, University of Sao Paulo Medical School, Sao Paulo, – Brazil. Tel.: 55-11-2661-7512; Fax: 55-11-2661-7519; or eutopic. No clear genotype phenotype correlation is obvious E-mail: [email protected] among the cases with HESX1 mutations, but there is a trend that

408 © 2016 John Wiley & Sons Ltd HESX1 mutations in patients with CPHD 409 the recessive mutations cause more severe phenotypes and appear Guidelines on the Practice of Ethical Committees in Medical to be fully penetrant while heterozygous mutations may be asso- Research, issued by the Royal College of Physicians of London. – ciated with milder phenotypes and reduced penetrance.6,9,10,12 14 Copies of the MRC recommendations can be obtained from the The approach of Sanger sequencing of candidate genes has led Medical Research Council. Patient 2Á2 was approved by the to the identification of most of the known genetic causes of National Research Ethics Commission (CONEP) and by the hypopituitarism. It is clear that hypopituitarism is a genetically Ethics Committee in Research (CEP) from the University of S~ao heterogeneous condition. The mutations in the reported genes Paulo, Medical School, S~ao Paulo, Brazil, functioning according account for less than 20% of the cases. Thus, candidate gene to the Resolution No. 466/2012 which deals with research and screening has a low detection rate. The recent introduction of testing in humans adopted by the Plenary of the National Health massive-parallel sequencing methods now offers the promise of Council (CNS) in 240ª ordinary meeting in December 2012. detecting mutations in known candidate genes, as well as the Exome sequencing of de-identified patient samples was approved identification of novel genes implicated in congenital hypopitu- by the IRBMED at University of Michigan. itarism. In this study, we report the identification of mutations in HESX1 in patients from three unrelated families with CPHD Exome sequencing and variant calling without midline defects or SODs. This is among the first examples of applying next-generation sequencing techniques to obtain Patient DNA samples from families 2 and 3 were subjected to a molecular diagnosis for hypopituitarism in humans.15,16 whole-exome sequencing. Exome capture was performed by the U-M Sequencing Core using the Nimblegen SeqCap EZ Human Exome Library v3.0 (Roche, Madison, WI, USA), targeting a Subjects and methods total of 64 Mb of the genome. Paired-end 100-base sequencing data were collected using an Illumina HiSeq2000 system. Exome Patients capture and sequencing for two patients, namely 2Á1 and 3Á2, Four patients from three unrelated families were recruited in were performed in the same batch. BWA v0.5.9 (http://bio-bwa.- this study (Fig. 1, Table 1). Two siblings (1Á1 and 1Á2) in family sourceforge.net/) was used to align Illumina reads to the 1000 1 were born to consanguineous parents of Middle-Eastern ori- Genomes Phase 1 reference mapped to GRCh37. Read pairs that gin. Patients 1Á1 and 1Á2 were initially diagnosed in the Middle mapped to multiple locations were removed; most of these loca- East and then treated at Great Ormond Street Hospital for Chil- tions contain highly repetitive sequences and are inaccessible to dren, London, UK. Patient 2Á2 in family 2 was born to a Brazil- short-read sequencing. PICARD v1.74 (Broad Institute, Cam- ian consanguineous family, and she was diagnosed and treated bridge, MA, USA) was used to remove duplicate read pairs. at the Clinical Hospital of the Faculty of Medicine of the Variant detection for both SNVs and small indels (<10 nt) was University of S~ao Paulo, S~ao Paulo, Brazil. Family 3 was diag- performed by the GATK Haplotype Caller v3.3. Multisample nosed and treated at Floating Hospital for Children at Tufts joint calling with 688 in-house exome samples was performed to Medical Center, Boston, MA, USA.17 remove the sequencing artefacts. Patient studies were approved by the ethical committees at each institution. Patients 1Á1 and 1Á2 were approved by a Sanger sequencing committee functioning according to the 3rd edition of the Sanger sequencing was used to analyse the HESX1 gene in patients from family 1. The variants identified in families 2 and 3 by exome sequencing were confirmed by Sanger sequencing.

SNP genotyping

To detect copy number variation, we performed genotyping on the two DNA samples for patients 2Á2 using Illumina’s Human OmniExpressExome_8v1_A at the University of Michigan Sequencing Core.

Plasmids

pCMV6-Entry-human HESX1 (Myc-DDK-tagged) was pur- chased from OriGene (Cat. No. RC210107, OriGene Technolo- gies, MD). The c.475C>T and c.479G>A changes were introduced into the HESX1 cDNA sequence using QuickChange Fig. 1 Pedigrees of families 1, 2 and 3. Filled symbols represent family II XL Site-Directed Mutagenesis Kit (Cat. No. 200521, Strata- members with pituitary hormone deﬁciency. Arrow points to the proband gene). The HESX1 cDNA sequences have been checked to con- in family 3. ﬁrm that except for c.475C>T and c.479G>A, no other

Table 1. Summary of selected clinical phenotypes and MRI ﬁndings of CPHD/SOD patients with HESX1 mutations

Patient Sex Clinical symptoms Affected hormones MRI ﬁndings HESX1 mutation Ref

Female and CPHD and SOD ACTH, GH, TSH, LH, FSH APH, EPP, ONH, hypoplasia p.R160C 6 and 9 male of the corpus callosum 1Á1 and 1Á2 Both females Hydrocephalus ACTH, TSH, GH APH/PA, NPP p.R160C This study Female Short stature ACTH, GH, TSH, LH, FSH APH, EPP p.I26T 11 2Á2 Female Short stature GH, TSH, LH, FSH APH, NPP p.I26T This study 3Á2 Male Lethargic and cyanotic ACTH, GH, TSH, LH, FSH PA, PP is present p.[R159W];[R160H] This study 8 h after birth and functioning

CPHD, combined pituitary hormone deﬁciency; SOD, septo-optic dysplasia; ONH, optic nerve hypoplasia; ACTH, adrenocorticotropin; FSH, follicle- stimulating hormone; GH, growth hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone; APH, anterior pituitary hypoplasia; PA, pituitary aplasia; PP, posterior pituitary; EPP, ectopic posterior pituitary; NPP, normal posterior pituitary.

mutations were incorporated. The pGL3-(P3)6E4 firefly Patient 2Á2 was the daughter of first-degree cousins. She first luciferase reporter, pcDNA3Á1(À)-human PROP1, has been presented with short stature (height 125Á9 cm, À6Á0 standard described and used previously.11 pcDNA3Á1(À) and pRL-TK deviation score (SDS)) at 17Á2 years of age, with a weight of renilla luciferase reporter vectors were from Invitrogen (Carls- 32Á6kg(1Á0 SD for stature age). Her bone age was delayed by bad, CA, USA) and Promega (Madison, WI, USA), respectively. 4 years. Her mid-parental height (MPH) was 150 cm (À2Á03 SDS). Clinical investigations revealed GH and gonadotropin deficiencies (peak GH <0Á1 ng/mL, FSH 1Á3 mU/mL, LH Cell culture and transfections 0Á7 mU/mL) and tertiary hypothyroidism (Free T4 0Á81 ng/dL, COS-7 cells were cultured in DMEM supplemented with 10% foe- basal TSH 5Á7 mU/mL with late response to TRH stimulation). tal bovine serum and 1% penicillin–streptomycin. Transient trans- She was treated with recombinant human GH and thyroxine. fections were carried out using FuGENE6 (Promega), following Puberty was induced at 21Á4 years old and her final height was the manufacturer’s protocol with modifications. Briefly, 1 9 105 151Á2cm(À1Á83 SDS), well within her target height range. She cells were seeded into each well on a 24-well plate 24 h before has not as yet developed ACTH deficiency. MRI of the pituitary transfection. Cells were transfected with 10 ng of pRL-TK renilla gland showed a normal stalk, anterior pituitary hypoplasia and a luciferase vector (Promega) to control for transfection efficiency eutopic posterior pituitary (Fig. 1, Table 1). 11 and 200 ng of (P3)6E4 firefly luciferase reporter. The DNA con- Patient 3Á2 was born to a nonconsanguineous pedigree with a centration of the other transfected plasmids varied depending on previously affected sister (Fig. 1). Detailed early clinical course of experimental protocol as indicated in the figure legends, but the this patient has previously been reported.17 Briefly, patient 3Á2isa total amount of DNA transfected per well was normalized to Caucasian boy. His older sister (3Á1) developed hypoglycaemia 400 ng by addition of the appropriate amount of empty expres- and died on the first day of life. A post-mortem examination sion vector. Cells were incubated and collected 48 h later and revealed absence of the anterior pituitary and atrophy of the adre- assayed for luciferase activity using the dual-luciferase protocol on nal glands. At 8 h of age, patient 3Á2 became lethargic and cyan- GLOMAX 96 Microplate Luminometer (Promega). otic and had a generalized seizure. Because of the similarity in presentation to that of his sister, a presumptive diagnosis of hypopituitarism was made, and he was treated with glucose and Results hydrocortisone. He was subsequently treated for hypothyroidism and growth hormone deficiency, and he achieved normal develop- Clinical characteristics mental milestones. He failed to develop secondary sexual charac- Patients 1Á1 and 1Á2 are consanguineous siblings who presented teristics as a teenager. His luteinizing hormone (LH), follicle- with hypoglycaemic seizures in the neonatal period and com- stimulating hormone (FSH) and testosterone concentrations plete ACTH, TSH, GH and prolactin deficiencies (undetectable remained in the prepubertal range on repeated tests. MRI con- cortisol, free thyroxine 2Á42 pmol/L in 1Á2, TSH <0Á005 in 1Á2, firmed absence of the anterior pituitary and presence of the poste- undetectable IGF1 and GH 0Á01 mU/L in 1Á2, and prolactin rior pituitary gland. His final height was 174 cm. Several years <11 mU/L in 1Á2). MRI revealed severe anterior pituitary hypo- later, at the age of 30 years, he died after developing severe gas- plasia (APH) and eutopic posterior pituitary. In both siblings, troenteritis with vomiting and diarrhoea (Fig. 1, Table 1). thyroxine and hydrocortisone were commenced in the neonatal period and growth hormone was commenced at the age of 1 Genetic analysis year. The older sibling had hydrocephalus and required pubertal induction at the age of 12 years. The younger sibling, currently Sanger sequencing was performed to identify the homozygous 6 years old, is too young to assess the hypothalamo–pituitary– p.R160C mutation in HESX1 in both patient 1Á1 and 1Á2 gonadal axis (Fig. 1, Table 1). (Fig. 1).

Whole-exome sequencing (WES) was performed on the geno- Functional studies mic DNA samples from patients 2Á2 and 3Á2. Alignment of the HESX1 acts as a transcriptional repressor by suppressing the reads and variant calling were performed as described in Meth- activity of PROP1.9,11,21 Mutations in either the homeodomain ods. For this study, we concentrated on potentially damaging or the engrailed homology (eh) domain of HESX1 impair this SNVs (nonsense, missense, stop loss, splicing change, frameshift, repressive ability.9,11 To test whether p.R159W and p.R160H etc.) and small indels (<10 nt). Filtering steps were made substitutions affect the repressive ability of HESX1, Cos-7 cells according to following criteria: reading depth of variants (≥10), were transiently transfected with plasmids expressing PROP1, minor allele frequency in ExAC, ESP and 1000G public data- normal HESX1 (HESX1-WT), HESX1-p.R159W and HESX1- bases (≤1% for homozygous variants and ≤3% for compound p.R160H. These expression vectors were cotransfected with the heterozygous variants), prediction that the variant has a deleteri- reporter construct (pGL3) containing 6 tandem paired homeo ous effect on the gene function by at least one software pro- domain consensus DNA binding sites (P3) upstream of the E4 gram, RVIS percentile (≤75%), (CADD Phred score (≥15) and 6 promoter activating the expression of firefly luciferase gene. As GERP++ score (≥4) (Fig. S1). Sanger sequencing was used to expected, PROP1 activated reporter gene expression, and confirm the candidate variants. HESX1-WT, HESX1-p.R159W and HESX1-p.R160H have no Because patient 2Á2 is from a consanguineous family, the most effect on transcription when tested individually (Fig. 2). When compelling variants are located in the runs of homozygosity equal amounts of HESX1-WT were cotransfected with PROP1, (ROH) regions and transmitted in a recessive inheritance pattern. PROP1 activation was repressed by ~50%. Neither HESX1- We identified two rare, homozygous variants in the ROH regions: p.R159W nor HESX1-p.R160H were able to repress PROP1 rs28936416 (c.77T>C, p.I26T) in HESX1 and rs141318879 activity. Transfection of equal amounts of HESX1-p.R159W and (c.888G>A, p.M296I) in HMGCLL1. A CPHD patient homozy- HESX1-p.R160H together with PROP1 was carried out to mimic gous for c.77T>C, p.I26T in HESX1 gene was previously reported, the compound heterozygous status of the HESX1 mutations in and the variant was shown to impair the transcriptional repression patient 3Á2. The combination of p.R159W and p.R160H was also properties of HESX1.11 HMGCLL1 gene encodes an isoenzyme of unable to repress PROP1 activation (Fig. 2a). This lack of human HMG-CoA lyase localized in the endoplasmic reticulum repression is not due to different expression levels of HESX1 (ER).18 There is no report on variants in HMGCLL1 gene causing proteins, as HESX1-WT, HESX1-p.R159W and HESX1-p.R160H hypopituitarism. Patient 2Á2 was also compound heterozygous for were expressed at comparable levels in the cells as determined six genes (ANGPTL1, EPHA1, CDCC88B, AGAP2, FASN, MBD1) by Western Blot analysis (Fig. 2b). which carry at least one allele passing all the filtering criteria, but none of them is located in the ROH regions of patient 2Á2’s genome. No variants were detected in other known genes for CPHD Discussion or IGHD. Therefore, HESX1 c.77T>C, p.I26T is the most likely pathogenic variant for the phenotype in patient 2Á2. Congenital hypopituitarism (CPHD) occurs in 1:4000 to 10 000 Patient 3Á2 was the second affected child in a family with non- births, and the molecular aetiology is unknown for the majority consanguineous parents. Therefore, we first considered a reces- of these patients, especially the sporadic cases.22 Mutations in sive inheritance pattern. We did not detect any homozygous HESX1 apparently account for 8% or less of CPHD cases.22,23 variants, but eight compound heterozygous variants were found Mutations were initially described in patients with SOD and in four genes on the autosomal chromosomes that passed later in patients presenting with nonsyndromic hypopitu- through the filtering steps. These four genes are HESX1, AK9, itarism.6,9,11,24 H6PD and CCDC168. Among them, only mutations in HESX1 In this study, we identified a homozygous HESX1 p.R160C are known to cause CPHD and/or SOD. The two variants we mutation by Sanger sequencing in two CPHD patients without found in HESX1 are c.475C>T, p.R159W and c.479G>A, SOD from a consanguineous Middle-Eastern pedigree. This p.R160H. We verified that the two variants are truly in trans p.R160C mutation was previously described in two siblings with using the Integrative Genomics Viewer (IGV) of individual CPHD and SOD from consanguineous patients of Pakistani ori- reads.19 Both p.R159W and p.R160H variants reside in the gin.6 The p.R160C change is located in the homeodomain, and homeobox domain of the HESX1 protein. A homozygous EMSA analysis showed that it abrogates DNA binding, consis- c.479G>A, p.R160H change, was previously reported to cause tent with loss of function.6,9 The discrepancy in the presence of CPHD.20 This is the first report of the variant c.475C>T, SOD in the two families with the same mutation suggests the p.R159W in a CPHD patient. The minor allele frequency (MAF) effects of other genes, environment or chance in enhancing or of the p.R159W change is less than 0Á002% (ExAC database), suppressing the severity of the phenotype. which means this variant is extremely rare in the general popu- We used exome sequencing to identify a homozygous p.I26T lation. All of the prediction software programs we used (SIFT, mutation in HESX1 in a Brazilian patient who was born to con- PolyPhen-2, Mutation Taster, Mutation Assessor and sanguineous parents and presented with CPHD and a eutopic FATHMM) predict that the p.R159W change has a deleterious posterior pituitary lobe. This phenotype differs from that previ- effect on HESX1 function. Thus, the compound heterozygous ously reported for a homozygous p.I26T mutation in an unre- variants p.[R159W];[R160H] are the most likely pathogenic lated Brazilian patient from consanguineous parents. That causes for the phenotype of patient 3Á2. patient had ACTH deficiency and an ectopic posterior pituitary

Fig. 2 Functional studies reveal failure of HESX1 compound heterozygous variants [p.R159W/ p.R160H] to repress PROP1 activity. (a) Plasmid constructs encoding human PROP1, HESX1-WT, HESX1-p.R159W and HESX1-p.R160H were transfected individually or in combinations into COS-7 cells to measure the activation of firefly luciferase reporter. HESX1-WT, HESX1-p.R159W and HESX1-p.R160H have no effect on transcription by themselves. Equal amounts of HESX1-WT repress PROP1 activation by ~50%. Neither HESX1-p.R159W nor HESX1-p.R160H were able to repress PROP1 activity. The combination of p.R159W and p.R160H was also unable to repress PROP1 activation. The results represent the means of three independent experiments, each performed in triplicate. (b) Western blotting (WB) was performed to show that p.R159W and p.R160H mutations do not affect the protein expression level of HESX1 in the COS-7 transfected cells. gland.11 The p.I26T mutation is located in the engrailed homol- anterior pituitaries. Telencephalic defects were detected in nearly ogy domain, which is required for recruitment of the corepres- 80% of Hesx1R160C/R160C mice, but not in Hesx1I26T/I26T mice. sor TLE1, and functional analysis revealed that the mutation Interestingly, neither Hesx1R160C/R160C nor Hesx1I26T/I26T mice decreases the repressive function of the protein.11,21 The muta- were deficient in the induction and differentiation of hormone- tion p.I26T has an allele frequency at 0Á002% in the general producing cells, although pituitary function could not be population and none in homozygous state (data from ExAC). assessed because of lethality.26 The phenotypic variability among We identified a compound heterozygous HESX1 mutation mice with the same genotype could be due to chance or the p.[R159W];[R160H] in a patient with CPHD, including aplastic action of epigenetic or environmental factors that affect how the anterior lobe but no SOD, using exome sequencing, and we mutations express themselves phenotypically.27 Alternatively, the demonstrated that each of these mutations impairs the repressive residual genetic variation in other genes and/or pathways may function of HESX1. In previous studies, two patients from dif- modify the severity of phenotypes.28 The completely sequenced ferent consanguineous families were reported to be homozygous genomes of inbred mouse strains and the international Collabo- for HESX1 p.R160H, but no functional studies were carried rative Cross (CC) project in mice would largely facilitate the out.20,25 Those patients presented with hypoplastic anterior pitu- mapping of the modifier genes. itary with deficiencies of GH, TSH, ACTH and prolactin. To explain the phenotypic variation in humans, we need more Genetically engineered mice provide an opportunity to assess comprehensive information about the patients’ phenomes and the variability in presentation among individuals of identical genomes. To discover genetic modifiers of a Mendelian trait by genotype through generation of large cohorts and the effects of WES, an extreme phenotype study design and/or large sample different genetic backgrounds by outcrossing to different inbred sizes are required to achieve the statistical power that is strains. A comparison of genetically engineered mice homozy- needed.29 WES provides the ability to detect potential disease gous for the Hesx1null, Hesx1R160C and Hesx1I26T mutant alleles causing variants in the coding regions across the genome. Given suggested that the p.R160C mutation is a null allele, and the that CPHD is a rare condition in the population and the known p.I26T is a hypomorph.26 Despite efforts to normalize the variants and genes only account for a minority of the cases, genetic background, there was some variability in presentation WES will improve the overall detection rate for CPHD muta- among animals with the same genotype. About 5% of homozy- tions. In a cohort of 23 unrelated CPHD patients currently gous Hesx1null mice have profound abnormalities that include undergoing WES at University of Michigan, only the 2 cases absent telencephalic vesicles, eyes, olfactory placodes and reported in this study were found to harbour pathogenic muta- Rathke’s pouches.6 The majority of Hesx1R160C/R160C and Hes- tions in a known gene. WES obviously offers the advantage of x1I26T/I26T mice had eye defects and enlarged and bifurcated identifying novel causes of CPHD and the potential for elucidat-

© 2016 John Wiley & Sons Ltd Clinical Endocrinology (2016), 85, 408–414 HESX1 mutations in patients with CPHD 413 ing multigenic mechanisms similar to those that have been ital pituitary hypoplasia and septo-optic dysplasia. Human observed in mice.28 Molecular Genetics, 10,39–45. 11 Carvalho, L.R., Woods, K.S., Mendonca, B.B. et al. (2003) A homozygous mutation in HESX1 is associated with evolving Acknowledgements hypopituitarism due to impaired repressor-corepressor interaction. The Journal of Clinical Investigation, 112, 1192–1201. We acknowledge Bob Lyons and the staff at the UM DNA 12 Sobrier, M.L., Maghnie, M., Vie-Luton, M.P. et al. (2006) Novel Sequencing Core for their contributions to the work. HESX1 mutations associated with a life-threatening neonatal phenotype, pituitary aplasia, but normally located posterior pitu- Funding itary and no optic nerve abnormalities. Journal of Clinical Endocrinology and Metabolism, 91, 4528–4536. Funding for this work was provided by the National Institutes 13 McNay, D.E., Turton, J.P., Kelberman, D. et al. (2007) HESX1 of Health (R01-HD030428 to SAC), Great Ormond Street mutations are an uncommon cause of septooptic dysplasia and Hospital Children’s Charity and Health Research Biomedical hypopituitarism. Journal of Clinical Endocrinology and Metabo- Research Centre at Great Ormond Street Hospital for Children lism, 92, 691–697. NHS Foundation Trust and University College London and 14 Vivenza, D., Godi, M., Faienza, M.F. et al. (2011) A novel Fundacß~ao de Amparo a Pesquisa do Estado de S~ao Paulo- HESX1 splice mutation causes isolated GH deficiency by inter- FAPESP (Process number: 2013/00073-8 to AFFB). fering with mRNA processing. European Journal of Endocrinology, 164, 705–713. 15 Webb, E.A., AlMutair, A., Kelberman, D. et al. (2013) ARNT2 Declaration of Interest mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies. Brain, 136, 3096–3105. 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