ethical approval was obtained from the university college london institute of child health / great ormond street hospital for children joint research ethics committee , and written consent was obtained from patients and/or parents .
dna was extracted from 2 patients with hypopituitarism and features of charge and their parents , if available .
an additional cohort of 100 patients with septo - optic dysplasia or hypopituitarism was screened ; 77 had septo - optic dysplasia with variable hormone deficiency ( 6 had ectopic posterior pituitary [ epp ] ) , 21 had combined pituitary hormone deficiency without additional midline defects ( 6 with epp ) , and 2 had isolated ghd ( normal posterior pituitary ) .
the coding region of chd7 consisting of 38 exons ( nm_017780 ) was sequenced in its entirety in all patients .
exons were amplified by pcr on an eppendorf thermocycler over 35 cycles with exon - flanking primers , designed using primer3 ( available at http://frodo.wi.mit.edu/primer3 ) .
pcr products were treated with microclean reagent ( catalog no . 2mcl-10 ; web scientific ltd . , crewe , united kingdom ) according to manufacturer 's instructions and then sequenced using bigdye v1.1 sequencing chemistry ( applied biosystems , foster city , california ) and analyzed on a 3730  1 dna analyzer ( catalog no . 625 - 0020 ; applied biosystems / hitachi , tokyo , japan ) .
for sequence variants identified , we screened 190 ethnically matched control alleles at the same loci and screened the chd7 database describing chd7 mutations / variants from over 800 patients ( www.chd7.org ) ( 3 ) , a control exome database of 1500 caucasian / african americans ( http://snp.gs.washington.edu/evs ) , and  1000 genomes  ( www.1000genomes.org ) .
two in silico splicing prediction models , human splicing finder , version 2.4.1 , and netgene2 ( www.cbs.dtu.dk/services/netgene2 ) , were used to assess the effect of splicing variants .
patients with chd7 variants were also screened for variants in hesx1 , sox3 , kal1 , prokr2 , fgfr1 , fgf8 , nelf , wdr11 , sox2 , and otx2 .
hormone measurements were performed in standard assays in the biochemistry laboratories of great ormond street hospital for children , kent and canterbury hospital ( uk ) and hospital universtari son dureta , palma de mallorca ( spain ) .
ethical approval was obtained from the university college london institute of child health / great ormond street hospital for children joint research ethics committee , and written consent was obtained from patients and/or parents .
dna was extracted from 2 patients with hypopituitarism and features of charge and their parents , if available .
an additional cohort of 100 patients with septo - optic dysplasia or hypopituitarism was screened ; 77 had septo - optic dysplasia with variable hormone deficiency ( 6 had ectopic posterior pituitary [ epp ] ) , 21 had combined pituitary hormone deficiency without additional midline defects ( 6 with epp ) , and 2 had isolated ghd ( normal posterior pituitary ) .
the coding region of chd7 consisting of 38 exons ( nm_017780 ) was sequenced in its entirety in all patients .
exons were amplified by pcr on an eppendorf thermocycler over 35 cycles with exon - flanking primers , designed using primer3 ( available at http://frodo.wi.mit.edu/primer3 ) .
pcr products were treated with microclean reagent ( catalog no . 2mcl-10 ; web scientific ltd . , crewe , united kingdom ) according to manufacturer 's instructions and then sequenced using bigdye v1.1 sequencing chemistry ( applied biosystems , foster city , california ) and analyzed on a 3730  1 dna analyzer ( catalog no . 625 - 0020 ; applied biosystems / hitachi , tokyo , japan ) .
details of the pcr conditions and primers are available upon request . for sequence variants identified , we screened 190 ethnically matched control alleles at the same loci and screened the chd7 database describing chd7 mutations / variants from over 800 patients ( www.chd7.org ) ( 3 ) , a control exome database of 1500 caucasian / african americans ( http://snp.gs.washington.edu/evs ) , and  1000 genomes  ( www.1000genomes.org ) .
two in silico splicing prediction models , human splicing finder , version 2.4.1 , and netgene2 ( www.cbs.dtu.dk/services/netgene2 ) , were used to assess the effect of splicing variants .
patients with chd7 variants were also screened for variants in hesx1 , sox3 , kal1 , prokr2 , fgfr1 , fgf8 , nelf , wdr11 , sox2 , and otx2 .
hormone measurements were performed in standard assays in the biochemistry laboratories of great ormond street hospital for children , kent and canterbury hospital ( uk ) and hospital universtari son dureta , palma de mallorca ( spain ) .
the first patient with features of charge syndrome and a chd7 variant was a caucasian male born at term with a birth weight of 3.72 kg ( + 0.34 sd score [ sds ] ) .
clinical features included tetralogy of fallot , sensorineural hearing loss , micropenis with bilateral undescended testes , abnormally shaped ears ( figure 2c ) , a squint and hypermetropia , trigonocephaly , a small occipital meningocele , and severe developmental delay .
he presented with short stature ( height , 4.9 sds ) at 7 years of age . developmental delay
he was classified as probable / possible charge using blake criteria ( 1 major , 5 minor criteria ) , but not as charge using verloes criteria ( 0 major , 5 minor criteria ) .
he was commenced on t4 and recombinant human gh ( rhgh ) , and his growth velocity increased to 22 cm / y ( figure 2b ) .
magnetic resonance imaging ( mri ) revealed a small anterior pituitary , an absent pituitary stalk with an undescended or epp at the tuber cinereum , and a thin corpus callosum ( figure 2a ) .
his mother , who carried the same chd7 variant , was short ( 152 cm ; 1.67 sds ) but had a normal igf - i concentration ( table 1 ) and had received t4 for primary hypothyroidism from the age of 11 ( tsh , 20
mu / l [ normal 0.45.0 mu / l ] ; free t4 , 9 pmol / l [ normal , 919
she did not manifest features of charge syndrome . the sister ( figure 2 , b and c ) ( birth weight , 4.060 kg [ + 2.65 sds ] at 37 wk gestation ) , who did not carry the chd7 variant , had an atrial septal defect , an abnormally shaped ear , a squint , and developmental delay , but no hearing loss . she was short ( height , 98.1 cm [ 2.92 sds ] at 5.75 y ) and had ghd but normal cortisol , prolactin , and free t4 concentrations ( table 1 ) .
her brain mri showed generalized white matter loss and midline abnormalities ( cavum septum pellucidum and vergae , small anterior pituitary , a single anterior cerebral artery , thin optic chiasm ) , but a normal infundibulum and posterior pituitary .
she currently has a normal height velocity of 8 cm / y at the chronological age of 7.59 years and has not yet been commenced on rhgh replacement .
a , mri in patient 1 revealed aph , an absent pituitary stalk ( aps ) with an undescended / epp at the tuber cinereum and a thin corpus callosum .
patient 1 presented with short stature at the age of 3 years and was commenced on gh at 7 years of age ( left arrow ) .
the sister of patient 1 was found to be gh deficient at 6 years of age .
c , photographs of patient 1 and his sister . left , patient 1 with probable / possible charge syndrome according to blake criteria .
right , the sister of patient 1 with an atrial septum defect ( aps ) , abnormal ear , squint , developmental delay , and ghd .
d , mri of patient 2 at 2 months ( left ) and 20 months of age ( right ) revealed aph and an aps with an epp at the tuber cinereum , bilateral colobomata , and underdeveloped frontal lobes .
patient 2 presented with short stature at the age of 3 years and has recently been commenced on gh treatment at the age of 3 years ( arrow ) .
phenotypes in patients with chd7 variations abbreviations : tshd , tsh deficiency ; fshd , fsh deficiency ; ft4 , free t4 ; sp , septum pellucidum ; cc , corpus callosum ; bl , bilateral ; hcg , human chorionic gonadotropin . endocrine deficits , charge features , and results of mri are shown in patients with chd7 variants .
the proband carries a novel heterozygous missense variant , c.2194c > g ( figure 1a ) , in exon 4 of chd7 , resulting in substitution of a highly conserved proline residue by alanine , p.p732a .
the variant was also detected in the unaffected mother , but not in the father or sister , in 1500 healthy caucasian / african americans ( http://snp.gs.washington.edu/evs ) , in  1000 genomes ,  or in 190 control alleles .
this variant is listed in the charge database ( http://www.chd7.org ) , but it represents the same english patient and his mother that we describe here ( prof . c. van ravenswaaij , university medical center groningen , personal communication ) .
the second patient is a caucasian male ( birth weight , 1.260 kg [ + 0.39 sds ] at 28.5 wk gestation ) , with a full spectrum of charge features ( coloboma , mild choanal atresia , deafness , abnormal ear shape , growth and developmental delay , genital hypoplasia ) .
he fulfilled both blake ( 3 major , 4 minor criteria ) and verloes criteria ( 2 major , 4 minor criteria ) for diagnosis of charge syndrome .
his endocrinopathy ( table 1 ) included ghd , with undetectable igf - i and a low igf binding protein 3 ( igfbp3 ) , gonadotropin deficiency ( undetectable lh and fsh , and undetectable t on 3-day human chorionic gonadotropin test ) , severe cortisol deficiency , and central hypothyroidism , but with a normal prolactin concentration .
mri at 2 months of age showed a thin corpus callosum and a cavum septum pellucidum , and at 20 months a small anterior pituitary and an absent pituitary stalk with an epp , located at the tuber cinereum ( figure 2d ) .
his inner ear structures and semicircular canals were normal ; however , his olfactory bulbs appeared small .
he was commenced on t4 , hydrocortisone , rhgh , and a 3-month course of i m t. his growth velocity increased from a pretreatment velocity of 6.6 cm / y to 15 cm / y ( figure 2e ) , and there was a marked improvement in phallus size .
genetic analysis revealed a novel heterozygous splice site variant in intron 35 , c.ivs35+6t>c ( figure 1b ) , in a region predicted to be important for splicing .
netgene2 showed a decrease ( 0.86 to 0.32 ) in the confidence with which the splice donor site will be detected .
the sequence variant was not identified in 190 control alleles , in the chd7 database ( www.chd7.org ) , in a control database of 1500 caucasian / african americans , or in  1000 genomes . 
no chd7 variants were identified in our septo - optic dysplasia / hypopituitarism ( without charge features ) cohort .
the first patient with features of charge syndrome and a chd7 variant was a caucasian male born at term with a birth weight of 3.72 kg ( + 0.34 sd score [ sds ] ) .
clinical features included tetralogy of fallot , sensorineural hearing loss , micropenis with bilateral undescended testes , abnormally shaped ears ( figure 2c ) , a squint and hypermetropia , trigonocephaly , a small occipital meningocele , and severe developmental delay .
he presented with short stature ( height , 4.9 sds ) at 7 years of age . developmental delay
he was classified as probable / possible charge using blake criteria ( 1 major , 5 minor criteria ) , but not as charge using verloes criteria ( 0 major , 5 minor criteria ) .
he was commenced on t4 and recombinant human gh ( rhgh ) , and his growth velocity increased to 22 cm / y ( figure 2b ) .
magnetic resonance imaging ( mri ) revealed a small anterior pituitary , an absent pituitary stalk with an undescended or epp at the tuber cinereum , and a thin corpus callosum ( figure 2a ) .
his mother , who carried the same chd7 variant , was short ( 152 cm ; 1.67 sds ) but had a normal igf - i concentration ( table 1 ) and had received t4 for primary hypothyroidism from the age of 11 ( tsh , 20
mu / l [ normal 0.45.0 mu / l ] ; free t4 , 9 pmol / l [ normal , 919
she did not manifest features of charge syndrome . the sister ( figure 2 , b and c ) ( birth weight , 4.060 kg [ + 2.65 sds ] at 37 wk gestation ) , who did not carry the chd7 variant , had an atrial septal defect , an abnormally shaped ear , a squint , and developmental delay , but no hearing loss . she was short ( height , 98.1 cm [ 2.92 sds ] at 5.75 y ) and had ghd but normal cortisol , prolactin , and free t4 concentrations ( table 1 ) .
her brain mri showed generalized white matter loss and midline abnormalities ( cavum septum pellucidum and vergae , small anterior pituitary , a single anterior cerebral artery , thin optic chiasm ) , but a normal infundibulum and posterior pituitary .
she currently has a normal height velocity of 8 cm / y at the chronological age of 7.59 years and has not yet been commenced on rhgh replacement .
a , mri in patient 1 revealed aph , an absent pituitary stalk ( aps ) with an undescended / epp at the tuber cinereum and a thin corpus callosum .
patient 1 presented with short stature at the age of 3 years and was commenced on gh at 7 years of age ( left arrow ) .
the sister of patient 1 was found to be gh deficient at 6 years of age .
c , photographs of patient 1 and his sister . left , patient 1 with probable / possible charge syndrome according to blake criteria .
right , the sister of patient 1 with an atrial septum defect ( aps ) , abnormal ear , squint , developmental delay , and ghd .
d , mri of patient 2 at 2 months ( left ) and 20 months of age ( right ) revealed aph and an aps with an epp at the tuber cinereum , bilateral colobomata , and underdeveloped frontal lobes .
patient 2 presented with short stature at the age of 3 years and has recently been commenced on gh treatment at the age of 3 years ( arrow ) .
phenotypes in patients with chd7 variations abbreviations : tshd , tsh deficiency ; fshd , fsh deficiency ; ft4 , free t4 ; sp , septum pellucidum ; cc , corpus callosum ; bl , bilateral ; hcg , human chorionic gonadotropin . endocrine deficits , charge features , and results of mri are shown in patients with chd7 variants .
the proband carries a novel heterozygous missense variant , c.2194c > g ( figure 1a ) , in exon 4 of chd7 , resulting in substitution of a highly conserved proline residue by alanine , p.p732a .
the variant was also detected in the unaffected mother , but not in the father or sister , in 1500 healthy caucasian / african americans ( http://snp.gs.washington.edu/evs ) , in  1000 genomes ,  or in 190 control alleles .
this variant is listed in the charge database ( http://www.chd7.org ) , but it represents the same english patient and his mother that we describe here ( prof . c. van ravenswaaij , university medical center groningen , personal communication ) .
the second patient is a caucasian male ( birth weight , 1.260 kg [ + 0.39 sds ] at 28.5 wk gestation ) , with a full spectrum of charge features ( coloboma , mild choanal atresia , deafness , abnormal ear shape , growth and developmental delay , genital hypoplasia ) .
he fulfilled both blake ( 3 major , 4 minor criteria ) and verloes criteria ( 2 major , 4 minor criteria ) for diagnosis of charge syndrome .
his endocrinopathy ( table 1 ) included ghd , with undetectable igf - i and a low igf binding protein 3 ( igfbp3 ) , gonadotropin deficiency ( undetectable lh and fsh , and undetectable t on 3-day human chorionic gonadotropin test ) , severe cortisol deficiency , and central hypothyroidism , but with a normal prolactin concentration .
mri at 2 months of age showed a thin corpus callosum and a cavum septum pellucidum , and at 20 months a small anterior pituitary and an absent pituitary stalk with an epp , located at the tuber cinereum ( figure 2d ) .
his inner ear structures and semicircular canals were normal ; however , his olfactory bulbs appeared small .
he was commenced on t4 , hydrocortisone , rhgh , and a 3-month course of i m t. his growth velocity increased from a pretreatment velocity of 6.6 cm / y to 15 cm / y ( figure 2e ) , and there was a marked improvement in phallus size . genetic analysis revealed a novel heterozygous splice site variant in intron 35 , c.ivs35+6t>c ( figure 1b ) , in a region predicted to be important for splicing .
netgene2 showed a decrease ( 0.86 to 0.32 ) in the confidence with which the splice donor site will be detected .
the sequence variant was not identified in 190 control alleles , in the chd7 database ( www.chd7.org ) , in a control database of 1500 caucasian / african americans , or in  1000 genomes . 
no chd7 variants were identified in our septo - optic dysplasia / hypopituitarism ( without charge features ) cohort .
charge syndrome is a continuum of multiorgan disorders of variable severity , often caused by chd7 mutations .
we describe 2 patients harboring rare sequence variants in chd7 , both of whom have features of charge syndrome accompanied by multiple pituitary hormone deficiencies and structural abnormalities of the pituitary gland .
charge syndrome is associated with hh and occasional ghd ; however , structural abnormalities of the pituitary gland have not previously been described .
although charge syndrome is classically associated with hh , our data suggest that in some patients , congenital hypopituitarism may be associated with structural abnormalities of the pituitary gland and variations in chd7 .
a previous study has suggested that ghd occurs in 9% of charge patients ; 3 patients with ghd in this study had aph ( 19 ) , showing that chd7 might have a role in anterior pituitary development .
one patient from a cohort ( n = 379 ) with a clinical diagnosis of charge has been reported to have tsh deficiency ( 1 ) .
acthd has been described in 1 patient with a clinical but not genetically confirmed diagnosis of charge syndrome ( 20 ) . to summarize , abnormalities of pituitary function including ghd , tsh deficiency , and acthd , with a small anterior pituitary size ,
have rarely been associated with charge syndrome , but an undescended / epp has not ( 19 , 21 ) , and we describe this association for the first time .
variations in other genes involved in pituitary development , for example hesx1 , can also result in ghd with aph and either a normal or epp .
our data support the hypothesis that chd7 variations may lead to a continuum of anatomical and functional hypothalamic / pituitary abnormalities .
most chd7 mutations are heterozygous de novo , with a small number of inherited cases usually from an unaffected parent ( 9 ) .
no genotype - phenotype correlation has been identified , but missense mutations are associated with a milder phenotype ( 4 ) . in both mice and humans ,
chd7/chd7 is expressed in the developing anterior pituitary , at lower levels in the intermediate and posterior lobes , and in the developing and mature hypothalamus ( 8) , suggesting a potential role of chd7 in the development of the hypothalamo - pituitary axis .
hurd et al ( 8) showed that homozygous mutant mice present with an olfactory pit and rathke 's pouch hypoplasia .
chd7 was identified as a sox2 transcriptional cofactor ; both genes physically interact and have overlapping genome - wide binding sites that regulate a set of target genes , such as gli2 ( 14 ) , which when mutated , can cause defects of pituitary development including an epp ( 22 ) .
chd7 and sox2 interaction may be further evidence of a potential role for chd7 in hypothalamo - pituitary development , given the role of sox2 here ( 23 ) .
layman et al ( 11 ) have shown a reduction in the expression of fgfr1 , bmp4 , and otx2 in the olfactory placode of chd7 mutant embryos , as well as a reduction of otx2 in the hypothalamus .
chd7 is also thought to be necessary for access to otx2 target genes in the olfactory epithelium ( 24 ) .
because mutations in otx2 in humans are associated with variable hypopituitarism , often with an undescended / epp , this could be a further mechanism whereby chd7 mutations could lead to structural abnormalities of the pituitary gland .
although chd7 mutations have been described in association with kallmann syndrome , usually additional charge features are present in kallmann syndrome patients with chd7 variants ( 25 ) .
our data parallel these findings ; the patients with congenital hypopituitarism without additional features of charge syndrome did not harbor chd7 variants , suggesting that features of charge may have to accompany the hypopituitary phenotype for chd7 to be implicated in the etiology . in patient 1 ,
the p.p732a variant occurs at a highly conserved amino acid , which is not part of a domain with an established function ( figure 1 , c and d ) .
the proband 's sister had abnormally shaped ears , abnormal facial features , and isolated ghd with a small anterior pituitary , but she did not carry the variant .
the mother , who carries the variant , does not have features of charge syndrome or hypopituitarism , although an mri to assess her pituitary anatomy could not be performed .
these data suggest that p.p732a may be a contributory factor to the etiology of the hypopituitary phenotype in our patient but is unlikely to be causative in isolation .
recent evidence suggesting that the genetic basis in many cases of kallmann syndrome is digenic / oligogenic could be extrapolated to include overlapping syndromes such as charge or congenital hypopituitarism .
a second mutation has not yet been identified in either patient , although we have screened other genes involved in hypothalamo - pituitary development and in kallmann syndrome ( 26 ) .
the ivs35 + 6t > c variant occurs in the intron / exon boundary of the exon encoding the first brk domain , suggesting that the final protein may be mistranslated , leading to disrupted brk and transcriptional activity mediated through the leucine zipper . because ivs35 + 6t > c is located in a proposed splicing region and is predicted to affect splicing using 2 in silico prediction models , it may cause transcription to proceed through into the intron , creating 63 new amino acids before an early stop codon . in this scenario , exons 3638
would not be translated , resulting in a truncated protein without a second brk domain or leucine zipper ( refer to figure 1d ) .
familial charge syndrome has been described , and in such families , offspring are often more severely affected than the parent ( 9 ) .
other family members occasionally seem to have milder associated clinical features without carrying the variant ( 27 ) , as noted in the sister of the proband in pedigree 1 , concluding that these features are unrelated to the variant in the proband and further supporting the possible presence of digenicity .
we have for the first time identified an epp in combination with severe hypopituitarism in 2 unrelated patients with variable features of charge syndrome and chd7 variants , which are of unknown significance .
our data suggest the need for pituitary function testing and mri in patients with charge syndrome
. however , chd7 mutations are not common in pituitary insufficiency or septo - optic dysplasia without features of charge .
we have for the first time identified an epp in combination with severe hypopituitarism in 2 unrelated patients with variable features of charge syndrome and chd7 variants , which are of unknown significance .
our data suggest the need for pituitary function testing and mri in patients with charge syndrome . however , chd7 mutations are not common in pituitary insufficiency or septo - optic dysplasia without features of charge .
 introduction : charge syndrome is a multisystem disorder that , in addition to kallmann syndrome / isolated hypogonadotrophic hypogonadism , has been associated with anterior pituitary hypoplasia ( aph ) . 
 however , structural abnormalities such as an ectopic posterior pituitary ( epp ) have not yet been described in such patients.objective:the aims of the study were : 1 ) to describe the association between charge syndrome and a structurally abnormal pituitary gland ; and 2 ) to investigate whether chd7 variants , which are identified in 65% of charge patients , are common in septo - optic dysplasia /hypopituitarism.methods : we describe 2 patients with features of charge and epp . 
 chd7 was sequenced in these and other patients with septo - optic dysplasia / hypopituitarism.results : epp , aph , and gh , tsh , and probable lh / fsh deficiency were present in 1 patient , and epp and aph with gh , tsh , lh / fsh , and acth deficiency were present in another patient , both of whom had features of charge syndrome . both had variations in chd7 that were novel and undetected in control cohorts or in the international database of charge patients , but were also present in their unaffected mothers . 
 no chd7 variants were detected in the patients with septo - optic dysplasia / hypopituitarism without additional charge features.conclusion:we report a novel association between charge syndrome and structural abnormalities of the pituitary gland in 2 patients with variations in chd7 that are of unknown significance . 
 however , chd7 mutations are an uncommon cause of septo - optic dysplasia or hypopituitarism . 
 our data suggest the need for evaluation of pituitary function / anatomy in patients with charge syndrome . 
Patients and Methods
Patients
Direct sequencing analysis
Hormone analysis
Results
Patient 1
Patient 2
Patients with hypopituitarism
Discussion
Conclusion