The morphogenesis of midline brain structures, eyes, optic nerves and optic tracts is governed by a cascade of transcription factors including SOX2, OTX2 and BMP4 1 . Congenital anophthalmia, which is among the most severe consequences of defects in this cascade, is often accompanied by pituitary dysfunction and growth failure due to growth hormone (GH) deficiency 2 3 4 5 6 . Mutations in SOX2 are the most common cause of anophthalmia, and 10% of their carriers also show growth retardation 1 . The co-occurrence of eye malformations and GH deficiency is the highest (30%) in patients with OTX2 mutations 3 . BMP4 defects can induce a similar brain and ocular phenotype 6 7 . Mutations ranging from single nucleotide substitutions to cytogenetically visible deletions have been reported in OTX2 and BMP4, which are both located in 14q22q23. We identified three unrelated patients with anophthalmia, partial hearing loss and pituitary defects due to microdeletions of this region. This gave us a unique opportunity to study not only the ocular phenotypes which are well associated with these rare genetic defects, but also other phenotypic features which are less well characterized.

Karyotyping did not show chromosome abnormalities in any of the three patients. Array CGH revealed interstitial deletions of 14q22q23 of varying size (Figure 2) in the absence of additional relevant submicroscopic aberrations. Patient 1 carried an 8.8 Mb long deletion (chr14:50293781–59068634, hg18) removing multiple genes including BMP4 and OTX2 but not the SIX gene cluster. Patient 2 had a deletion of 8.9 Mb (chr14:54251697–63177878) affecting OTX2 and the SIX gene cluster. Patient 3 had a 5.8 Mb long deletion (chr14:54431790–60167626) removing OTX2 and a part of the SIX gene cluster. Using independent methods, all deletions were confirmed in the patients but not in any of the parents, thus indicating the de novo nature of the aberrations.

We present a case series of three patients with bilateral anophthalmia caused by microdeletions of 14q22q23. Their phenotype was further characterized by hearing impairment, abnormal pituitary development leading to GH deficiency and early growth failure, and dysmorphic facial features. The overview of phenotypes observed in published cases with 14q22q23 deletions and in our three patients is in Table 1.

N.D., not described; N.A., not applicable; *patient too young; ^#not specified if secondary (central) or primary (peripheral).

Similarly to Figure 2, this table does not list patients with deletions assessed using karyotyping in whom the genes affected are uncertain and small deletions affecting only OTX2 or BMP4 exons. The deletions in patients described by Bakrania et al., 2008 were identified using karyotyping and MLPA analysis was used to show that both deletions affected both OTX2 and BMP4.

The 14q22q23 region is critical for eye and pituitary development. Anophthalmia and other ocular anomalies were associated with heterozygous defects in OTX2 3 8 9 or BMP4 7 10 11 , and also with deletions involving both these genes 4 6 12 . While OTX2 was deleted in all our patients, BMP4 was deleted only in Patient 1 (Figure 2). Nevertheless, the ocular phenotype was similar in all three children. The phenotypic effect of OTX2 and BMP4 disruptions is very variable ranging between anophthalmia/microphthalmia, corneal opacity and no abnormality at all, even in family members with the same mutation 2 5 9 10 11 12 13 , and the phenotype does not have to be more severe in patients with combined OTX2/BMP4 defects 4 12 . Patients 2 and 3 also lacked SIX6; however, defects of this candidate gene have not been identified in anophthalmia 1 .

All three patients suffered from transient partial conductive hearing loss. Previous reports of patients with 14q22q23 deletions were inconsistent, varying from not mentioning the hearing status over normal function 7 12 to severe unilateral hearing loss 4 9 , indicating very variable expressivity. We speculate that skeletal abnormalities of the facial-cranial junction of the skull associated with anophthalmia due to the OTX2 defect can induce stenosis of ear canals and/or Eustachian tubes and hearing impairment. Changing proportions of these structures during growth could also explain the gradual improvement of hearing with age observed in our patients. Interestingly, Patient 2 with large low-set dysplastic ears was the only our patient with SIX1 disruption. Homozygous knockout of this gene in mice causes malformations of the auditory system including outer ears 14 . Deletions in two published patients also involved SIX1 and were associated with malformed ears, although differently from Patient 2 4 9 . OTX2 defects themselves could also contribute to ear anomalies 15 .

Our patients showed abnormal pituitary development, GH deficiency and growth retardation. Normal birth parameters and postnatal growth failure similar to that in Patients 1 and 2 were reported in some cases with 14q22q23 deletions 3 4 11 12 . On the other hand, Patient 3 suffered from severe intrauterine growth retardation without postnatal catch-up. An improvement of growth was evident after GH therapy in all our patients although the response varied. In Patients 1 and 3 the therapy induced an increase of growth velocity and improvement of their height, whereas in Patient 2 it just prevented further growth deterioration. However, in Patient 2 the growth failure was least pronounced with height of −1.8 SD at the start of the GH therapy, and this fact could influence the GH response. Similarly, two published 14q22q23 deletion patients treated with GH remained with their height at −2 SD after five and three years of therapy 4 13 , and the height of a boy with a missense OTX2 mutation remained at −2.3 SD after eight years of therapy 2 . As no reports are currently available on final height of 14q22q23 deletion patients, it remains to be seen if Patients 1 and 3 correct their growth failure completely or if they just reach the current height range of Patient 2. Differences in the deleted genes do not offer an obvious explanation for the differences in responsiveness to the GH therapy. Two genes involved in pituitary development, OTX2 and SIX6, were disrupted in Patients 2 and 3. Patient 1 had a deletion of OTX2 and BMP4, but not of SIX6. The differences in pituitary morphology between Patient 1 and Patients 2 and 3 can also be attributed solely to the variability seen among carriers of isolated OTX2 defects 1 3 .

Finally, polydactyly was present in Patient 1 with a deletion of BMP4. This gene plays an important role in the onset of endochondral bone formation in humans, and its mutations were associated with polysyndactyly 4 11 .

Recently, two papers were published describing patients with deletions overlapping the proximal and distal part of the 14q22q23 region 16 17 . A family with Frias syndrome carried a deletion of 14q22.1q22.3 spanning the interval between GNG2 and KTN1, with BMP4 haploinsufficiency being likely responsible for the phenotype which included hypoplasia of corpus callosum, minor ocular anomalies, specific tooth defects, digit anomalies, short stature and intellectual impairment 16 . The other deletion involving 14q22.3q23.2 and extending from PSMA3 over the SIX cluster to SYNE2 was identified in a patient with facial dysmorphism, choanal atresia, esophageal reflux, defects of hands and feet, seizures and intellectual disability 17 . Interestingly, in the latter case neither microphthalmia/anophthalmia nor pituitary anomalies were present (MRI was normal), and neither OTX2 nor BMP4 were deleted. These papers further illustrate the intra- and interfamilial variability which, together with biased and incomplete reporting of the phenotypes, complicates the genotype-phenotype correlation in patients with unique deletions affecting multiple genes which participate in different molecular pathways 16 17 .

Our case series study of three patients with deletions of 14q22q23 demonstrated the phenotypic features and variable expressivity of this genetic defect. Comparison with previously published patients with similar microdeletions and mutations in the OTX2 gene suggests that most symptoms presented by affected patients could be attributed to OTX2 haploinsufficiency. Growth retardation due to GH deficiency is very remarkable in these patients and GH treatment can increase the growth velocity especially in the most severe cases but the response can be atypical. The hearing impairment can be transient and improves with age.

Written informed consent was obtained from the parents of the patients for publication of this report and the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

The authors declare that they have no competing interests.

SB, ZSl, PD, ABS, CM, LT and MG carried out the cytogenetic and molecular cytogenetic studies; JL, ZSu, MS, EZ, SB, CM, KM and PL participated in the analysis of the phenotype and genetic counseling; PD, ZSe, JL and SB drafted the manuscript; ZSu edited the manuscript; ZSe, JL and GT conceived, designed and coordinated the study. All authors read and approved the final manuscript.