Clinical and mutation analysis of 51 probands with anophthalmia and/or severe microphthalmia ...

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Year: 2013
Clinical and mutation analysis of 51 probands with anophthalmia and/or
severe microphthalmia from a single center
Gerth-Kahlert, C ; Williamson, K ; Ansari, M ; Rainger, J K ; Hingst, V ; Zimmermann, T ; Tech, S ;
Guthoff, R F ; van Heyningen, V ; FitzPatrick, D R
Abstract: Clinical evaluation and mutation analysis was performed in 51 consecutive probands with
severe eye malformations – anophthalmia and/or severe microphthalmia – seen in a single specialist
ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions
ofSOX2, OTX2, PAX6(paireddomain), STRA6, BMP4, SMOC1, FOXE3, andRAX,andgenome-wide
array-based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations
affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia,
9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions
encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly
pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child
with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder.
Two families with SOX2 mutations demonstrated a parent and child both with significant but highly
variable eye malformations. Heterozygous loss-of-function mutations in SOX2 and OTX2 are the most
common genetic pathology associated with severe eye malformations and bi-allelic loss-of-function in
STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations.
DOI: https://doi.org/10.1002/mgg3.2
Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-79338
Journal Article
Published Version

The following work is licensed under a Creative Commons: Attribution 3.0 Unported (CC BY 3.0)
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Originally published at:
Gerth-Kahlert,C;Williamson,K;Ansari,M;Rainger,JK;Hingst,V;Zimmermann,T;Tech,S;Guthoff,
R F; van Heyningen, V; FitzPatrick, D R (2013). Clinical and mutation analysis of 51 probands with
anophthalmiaand/orseveremicrophthalmiafromasinglecenter. MolecularGenetics GenomicMedicine,
1(1):15-31.
DOI: https://doi.org/10.1002/mgg3.2
Clinical and mutation analysis of 51 probands with
anophthalmia and/or severe microphthalmia from a
single center
Christina Gerth-Kahlert1, Kathleen Williamson2, Morad Ansari2, Jacqueline K. Rainger2,
Volker Hingst3, Theodor Zimmermann4, Stefani Tech1, Rudolf F. Guthoff1,
Veronica van Heyningen2 & David R. FitzPatrick2
1DepartmentofOphthalmology,UniversityofRostock,Germany
2MRCHumanGeneticsUnit,MRCInstituteofGeneticsandMolecularMedicineattheUniversityofEdinburgh,WesternGeneralHospital,
Edinburgh,EH42XU,UnitedKingdom
3DepartmentofRadiology,UniversityofRostock,Germany
4DepartmentofPediatrics,UniversityofErlangen,Germany
Keywords Abstract
Anophthalmia,arrayCGH,BMP4,coloboma,
denovomutations,FOXE3,genedeletion, Clinical evaluation and mutation analysis was performed in 51 consecutive pro-
haploinsufficiency,microphthalmia,missense bands with severe eye malformations – anophthalmia and/or severe microph-
mutations,OTX2,PAX6,RAX,SMOC1, thalmia – seen in a single specialist ophthalmology center. The mutation
SOX2,STRA6,transcriptionfactors. analysis consisted of bidirectional sequencing of the coding regions of SOX2,
OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and
Correspondence
genome-wide array-based copy number assessment. Fifteen (29.4%) of the 51
DavidR.FitzPatrick,MRCHumanGenetics
probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51),
Unit,MRCInstituteofGeneticsand
and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were
MolecularMedicineattheUniversityof
Edinburgh,WesternGeneralHospital, found to be mutation positive. Three of these mutations were large genomic
Edinburgh,EH42XU,UnitedKingdom. deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheri-
Tel:[phone];Fax:[phone] tance of three intragenic, plausibly pathogenic, and heterozygous mutations was
8456;E-mail:david.fi[email protected] observed. An unaffected carrier parent of an affected child with an identified
OTX2 mutation confirmed the previously reported nonpenetrance for this dis-
FundingInformation
order. Two families with SOX2 mutations demonstrated a parent and child
Nofundinginformationprovided.
both with significant but highly variable eye malformations. Heterozygous loss-
Received:6December2012;Revised:26 of-function mutations in SOX2 and OTX2 are the most common genetic
January2013;Accepted:29January2013 pathology associated with severe eye malformations and bi-allelic loss-of-
function in STRA6 is confirmed as an emerging cause of nonsyndromal eye
MolecularGenetics&GenomicMedicine
malformations.
2013;1(1):15–31
doi:10.1002/mgg3.2
magnetic resonance imaging (MRI) or ultrasonography
Introduction
(for an example see Fig. 3). Microphthalmia can be
Severeeyemalformationsareaclinicallyimportantbutrare defined as an eye with an axial length of <19 mm in a
group of developmental disorders with a combined live 1-year-old child or <21 mm in an adult (2 SD below nor-
birth prevalence of 1 per 10,000 (Shah et al. 2011, 2012). mal; Weiss et al. 1989a,b). Microphthalmia can occur
Anophthalmia is the absence of the globe in presence of without (simple type) or with anterior and or posterior
ocular adnexa. An extremely rare subgroup is termed segment dysgenesis (complex type). Ocular coloboma,
true anophthalmia, which is associated with the complete complex microphthalmia, and both clinical and true
absence of neuroectodermal tissue in the orbit. The more anophthalmiaprobablypresentacontinuumofmalforma-
common presentation is termed “clinical anophthalmia.” tionseverity.
In this form, no ocular tissue is visible on examination Anophthalmiaandcomplexmicrophthalmiaareetiolog-
(Duke-Elder1964)butremnantsoftheopticvesicle, mus- ically heterogeneous. Causative mutations are identified in
cle or optic nerve tissue or cysts are detected by orbital many different genes including SOX2 (Fantes et al. 2003),
ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc. 15
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,
distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.
GeneticCausesofAnophthalmia C.Gerth-Kahlertetal.
OTX2 (Ragge et al. 2005a), PAX6 (Glaser et al. 1994), sity of Rostock (reference A 2008-62) and the U.K. Multi-
STRA6 (Pasutto et al. 2007), FOXE3 (Reis et al. 2010), regionalEthicsCommittee(Reference:06/MRE00/76).
RAX (Voronina et al. 2004; Lequeux et al. 2008), SMOC1
(Abouzeid et al. 2011; Okada et al. 2011; Rainger et al.
Phenotype assessment
2011), and BMP4 (Bakrania et al. 2008). Heterozygous
loss-of-function mutations in either SOX2 account for Allpatientsandavailableparentsreceivedacomprehensive
about 15% of cases with severe bilateral eye developmental eye examination including dilated fundus examination.
defects (Bardakjian et al. 1993; FitzPatrick 1993; Ragge Birth history, extraocular anomalies, and developmental
et al. 2005b) or OTX2 mutations ranging between 2% and data with emphasis on speech and motor development
8% of the investigated cohort (Ragge et al. 2005a; Wyatt were extracted from medical reports and parent interview.
et al. 2008; Schilter et al. 2011). Heterozygous, loss-of- Sleep abnormalities were evaluated using the sleep distur-
function mutations in the OTX2 gene commonly occur de bance scale for children through parent interviewing
novoinaffectedchildrenbutin35%ofcasesthemutation (Eitner et al. 2007). Z-scores for height, weight, and head
is inherited from a completely normal parent (Ragge et al. circumference were calculated using the LMS method
2005a;Schilteret al.2011).SOX2mutationsalmostalways based on data from U.K. children (Cole et al. 1998).
occur de novo and appear to be fully penetrant with all Cerebral MRI was reassessed if available. The analysis
reports of sibling recurrence associated with detectable of intracerebral abnormalities was focused on corpus
mosaicism in the mother (Faivre et al. 2006; Chassaing callosum, hypothalamus, and hippocampus integrity
et al.2007;Schneideret al.2008). based on published data of patients with microphthalmia
Here, we describe a clinical evaluation and comprehen- or anophthalmia (Leichtman et al. 1994; Warburg et al.
sive mutation analysis in 51 probands with microphthal- 1997; Arnold et al. 2003; Ragge et al. 2005a,b; Makhoul
mia and/or anophthalmia seen in a single specialist et al. 2007; Kelberman et al. 2008; Schneider et al. 2008;
ophthalmology center. Genome-wide oligonucleotide Tajima et al. 2009). Agenesis of the corpus callosum
array-based comparative genomic hybridization (aCGH) refers to complete absence whereas hypoplasia refers to a
was performed on each proband together with sequencing fully formed corpus callosum that is thinner than
of the coding regions of SOX2, OTX2, PAX6 (paired expected for age. Results of previous image evaluation
domain), STRA6, BMP4, SMOC1, FOXE3, and RAX. We were included in the study if original MRIs were not
identified different mutations in the 15/51 (29.4%) of the available.
probands in SOX2 (9/51), OTX2 (5/51), and STRA6 (1/
51).The mutation-positive individuals were morelikelyto
Intragenic mutation analysis
have bilateral rather than unilateral eye disease (13:3).
Indeed 9/12 (75%) of the cases with bilateral anophthal- Genomic DNA was whole genome amplified using
mia (BA) were found to be mutation positive. Familial GenomiPhi V2 DNA Amplification Kit (GE Healthcare
inheritance of a plausibly pathogenic heterozygous muta- Life Sciences, Pittsburg, PA) prior to mutation screening.
tion was seen for both SOX2 (two families) and OTX2 Toensureacompletedataset,alleightgeneswerescreened
(one family). In the latter family, an unaffected carrier in all affected individuals regardless of whether a plausibly
parent of an affected child with an identified OTX2 muta- pathogenic mutation had been detected. Identified muta-
tion was consistent with the previously reported nonpene- tions were validated using genomic DNA that had not
trance for this disorder. For SOX2, both families been whole genome amplified. For each gene, the ampli-
demonstratedfullpenetrancebutvariableexpressivitywith cons encompassing the coding exons and the intronic
parents and children associated with SOX2 mutation each splice junctions were designed using ExonPrimer or by
havingsignificanteyemalformations. manual means. Full methods and oligonucleotide
sequences are available on request. Each amplicon was
sequenced on the ABI 3730 DNA Analyzer (Applied Bio-
Methods
systems, Foster City, CA) and the results were analyzed
withMutationSurveyorv3.30(SoftGenetics,StateCollege,
Case ascertainment
PA). The genomic sequence identifiers for each gene ana-
Patients and parents of children with unilateral anopht- lyzed are as follows: SOX2, NT_005612.15 GI:88966845;
almia (UA) or BA or microphthalmia referred to the OTX2, NT_026437.11 GI:51493278; PAX6, NC_000011.9
Department of Ophthalmology at the University of Ro- GI:224589802;STRA6,NG_009207.1GI:219275560;BMP4,
stock for evaluation of surgical therapy with orbital or NG_009215.1 GI:219521814; SMOC1, NC_000014.8 GI:
socket expander were invited to participate in this study. 224589805;FOXE3,NG_016192.1GI:281182531;andRAX,
ThestudywasapprovedbytheEthicsboardoftheUniver- NG_013031.1GI:260655998.
16 ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc.
C.Gerth-Kahlertetal. GeneticCausesofAnophthalmia
In silico analysis elsewhere (Weier et al. 1991). The BAC clone RP11-
43F17, which spans SOX2, was selected from the UCSC
Assessment of the pathogenic potential of the missense
Human Genome Browser (http://genome.ucsc.edu) and
mutations used different bioinformatics approaches. Poly-
the probe was labeled with biotin-16-dUTP (Roche, Basel,
Phen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://
Switzerland) by nick translation (Fantes et al., 2008). Fol-
sift.jcvi.org), and ALIGN-GVGD (http://agvgd.iarc.fr/
lowing hybridization, slides were mounted with a drop of
agvgd_input.php) use different algorithms to produce a
Vectorshield antifadent containing DAPI (Sigma Aldrich,
combined score based on a multispecies alignment and
St Louis, MO). Antibody detection was carried out by
the biophysical characteristics of the altered amino acids
fluorescent microscopy using a Zeiss Axioscop micro-
(Tavtigian et al. 2006; Kumar et al. 2009; Adzhubei et al.
scope.ImageswerecollectedusingacooledCCD(charged
2010). Mutation Taster (http://www.mutationtaster.org/)
coupleddevice)camera(Smart-Capturesoftware).
uses a Bayes Classifier that was trained on large sets of
pathological and benign amino acid substitutions. The
possible effect of one of the missense mutation on splice Results
site function used different prediction algorithms on the
mutant and wild-type allele implemented online using Summary of cohort and mutation analysis
SSF (http://www.umd.be/searchSpliceSite.html), MaxEnt results
(Yeo and Burge 2004), and HSF (http://www.umd.be/
Fifty-one unrelated probands were enrolled in the study.
HSF/). These algorithms were implemented via the Alamut
Inclusion of mutation-positive parents gives a total of 54
package (Interactive Biosoftware, San Diego, CA). The
individuals (Table 1). One of these carrier parents did
haploinsufficiency (HI) score was calculated manually
not show any ocular abnormalities but is included as a
using the BED format dataset from the original report
“case.” The male:female ratio was 24:30. Of the 53
(Huang et al. 2010) loaded as a custom track into the
affected cases, 42 had anophthalmia (unilateral:bilat-
UCSC Genome Browser (http://genome.ucsc.edu/).
eral = 30:12), 10 had severe microphthalmia (unilateral:
bilateral = 7:3), one (a mutation-positive parent) had
Array-based comparative genomic bilateral coloboma.
hybridization No plausibly pathogenic mutations or copy number
variants were identified in 36 cases. Of the 18 mutation-
Genome-wide analysis of DNA copy number aberrations positive cases, one had bi-allelic intragenic compound
was carried out using the Roche Nimblegen 12X135k heterozygous loss-of-function mutations in STRA6. Eleven
whole-genome array (median probe spacing of approxi- cases from nine families had heterozygous loss-of-func-
mately 12 kb) according to the manufacturer’s instruc- tions mutations affecting SOX2. Of these nine mutations,
tions, modified as follows: 250–800 ng of genomic DNA two were transmitted from mother to child. In both these
frompatientandsex-matchedcontrolsamples(pooloffive families, analysis of the grandparental samples confirmed
maleorfemalesamples)waslabeledovernightfollowedby that the mutation had occurred de novo in the mother.
hybridization for 72 h. After washing, slides were scanned Of the seven remaining SOX2 mutations, three occurred
using the Roche MS200 scanner, and analyzed using the de novo and four families did not have samples from
software NimbleScan (Roche Nimblegen, Madison, WI). both parents available for analysis (Table 2).
The CGH-segMNT module of NimbleScan was used for Six cases from five families had heterozygous loss-
the analysis with a minimum segment length of five of-function mutations affecting OTX2. One of these
probes and an averaging window of 130 kb. Results were mutations was transmitted from mother to child. One
compared with the Database of Genomic Variants (http:// mutation occurred de novo in the affected individual and
projects.tcag.ca/variations) and polymorphic copy number in the remaining three families samples from both parents
variation excluded. Deletions of the OTX2 gene were con- were not available to determine inherited status. None of
firmed using the Roche Nimblegen 3X720k whole-genome the mutation-positive cases has associated facial clefts,
array (median probe spacing of approximately 2.5 kb) as heart, or kidney anomalies (Table 3).
described above, with an averaging window of 20 kb.
Segmental aneuploidy cases
Fluorescence in situ hybridization
SOX2 HI in CaseID 2850 resulted from a de novo 1.6Mb
Deletion of the SOX2 gene was confirmed by fluorescence genomic deletion (chr3:181,166,306–182,856,306; hg19)
in situ hybridization (FISH). Metaphase chromosomes (Fig. 1). This deletion encompasses six genes in addition
were prepared from the patient lymphocytes as described to SOX2 (including SOX2OT). This child has BA but sur-
ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc. 17
GeneticCausesofAnophthalmia C.Gerth-Kahlertetal.
Table1. Phenotypecomparisonofpatientswithandwithoutidentifiedmutations.
Nomutation Molecular
identified OTX2mutation SOX2mutation STRA6mutation Total diagnosticrate
Cases(families) 36(36) 6(5) 11(9) 1(1) 54(51) 33.3(29.4)
Male:Female 16:20 2:4 5:6 1:0 24:30 33.3:33.3
Ocularphenotype
Bilateralanophthalmia 3 3 5 1 12 75.0
Unilateralanophthalmia 23 2 52 0 30 23.3
Bilateralmicrophthalmia 3 0 0 0 3 0.0
Unilateralmicrophthalmia 7 0 0 0 7 0.0
Bilateraliris/chorio-retinalcoloboma 0 0 12 0 12
Unaffected 0 12 0 0 12
Nolightperceptioninbotheyes1 6 4 7 1 18 66.7
Extraocularabnormalities
IntracerebralMRI(excludingoptictract) 11/213 1/5 6/8 0/1
Facialclefts 6 0 0 0
Extremities 3 1 0 0
Earabnormalities 5 1 0 0
Heart 5 0 0 0
Kidney 3 0 0 0
MRI,magneticresonanceimaging.
1Atmostrecentvisit.
2Oneparent(mother)affected.
3Availabledata.
prisingly had normal development with no significant (MIM# 606326 Microphthalmia with cataract 2 – dis-
extraocular features apart from a pineal cyst detected on cussed below), SIX1 (MIM# 601205 Brachiootic syndrome
MRI at 4.5 years (Table 2). This suggests that the other 3 and deafness, autosomal dominant 23), and PRKCH
deleted genes have little or no haploinsufficient effect. (MIM# 605437 susceptibility to cerebral infarction). The
The HI score is designed to predict a haploinsufficient mean HI score of these genes is 40.5% and ranges from
effect of an individual gene. It is a combined metric 0.5% (PPM1A) to 98.2% (C14orf101). This individual had
incorporating evolutionary conservation of the open read- BA with significant developmental delay (Table 3). Brain-
ing frame (ORF) and promoter, embryonic expression stem auditory evoked response (BAER) recording at age
and proximity to known haploinsufficient genes in pro- 2 weeks and 3 months revealed normal responses between
tein–protein and probabilistic gene interaction networks 80 and 60 dB. No reliable response could be documented
(Huang et al. 2010). The mean HI score of the genes in at 50 dB. She wore hearing aids till age 2 then she refused
the deletion in CaseID 2850 is 35.5% and ranges from to wear them. Repeated BAER recordings at age 2.5 years
4.6%.(SOX2) to 69.7% (LAMP3). showed good responses from 60 to 30 dB, which was con-
Thiswasduetoadeletionintwootheraffectedindivid- firmed at later age. The aCGH and FISH data for each of
uals with OTX2 heterozygous loss-of-function (Fig. 1). thewholegenedeletionmutationsareshowninFigureS1.
CaseID 3346 has a de novo 455Kb deletion (chr14:57,
154,247–57,609,247; hg19) which encompasses only OTX2
and thenoncoding OTX2-AS1 gene. This child is develop- SOX2 mutation cases
ing well with normal speech (he started to speak at age
Consequence of mutations
8 months, loves to sing) and motor development. Now at
age2.6 years,hewalksinareasknowntohimwithoutany Of the eight intragenic mutations in SOX2, five would
problems, even up and down stairs. His otoacoustic emis- plausibly result in complete loss-of-function, four frame-
sions (OAEs) and auditory brainstem responses (ABR) at shiftmutations,andonenonsensemutation(Tables 2and
age 8 months indicate a normal hearing level in both ears 4). The remaining three mutations consisted of two mis-
(Table 3). CaseID 3000 has a 6.5 Mb deletion sense changes, p.(His101Arg) and p.(Asp123Gly), and one
(chr14:57,024,247–63,524,247; hg19), however, both in-frame duplication, p.(Ala47dup). Residues Ala47 and
parental samples were not available to determine de novo His101liewithin theHMGdomain ofSOX2 and both are
status (Fig. 1). This deletion encompassed 36 genes in completely evolutionarily conserved to Drosophila mela-
additiontoOTX2,includingtheknowndiseasegenesSIX6 nogaster SoxNeuro protein. The HMG domain is known
18 ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc.
C.Gerth-Kahlertetal. GeneticCausesofAnophthalmia
to make direct contact with DNA, to bend the DNA and weight were −2.92, −0.85, and −1.16. Developmental
strand, and to mediate protein–protein interactions with follow-up information was available in 8/10 affected indi-
the cooperative binding partner transcription factor when viduals: four and seven cases had delayed speech and
bothareboundtothetargetsite.ResidueAla47lieswithin motor development, respectively. None of the individuals
the first alpha helix of the HMG domain. It is adjacent to with SOX2 intragenic mutations show any significant
Asn46 which directly contacts DNA (Remenyi et al. 2003) extracranial malformations or abnormalities.
and is within a cluster of amino acids that mediate the Brain MRI was available in 7/10 cases with intragenic
bending of the DNA strand (Scaffidi and Bianchi 2001). mutations and one individual with a deletion. Of these
Residue His101 lies immediately C-terminal of the third eight cases, six had reported abnormalities on the scan.
alpha helical region but no specific role in the known Four of eight cases had abnormal or dysmorphic midline
HMG functions hasbeen defined for this residue. Aspartic structure such as hypoplasic posterior corpus callosum,
acid 123 (Asp123) lies outside the HMG domain in a septum pellucidum cyst, or cavum vergae. Fronto or fron-
region of unknown function but this residue is completely to-temporal cerebral volume reduction but normally
conserved within sequences from vertebrate species. The developed corpus callosum and midline structures were
identical nucleotide change causing the p.(Asp123Gly) diagnosed in two of eight imaged children (Fig. 3).
mutation here has been previously reported in a four-gen-
eration family with variable eye malformation (Mihelec
et al. 2009). These are clearly independent events as the OTX2 mutation cases
mutation in FamID 3227 has occurred de novo in the
Consequence of mutations
affected mother. The chromatograms and pedigrees for
eachoftheintragenicmutationsareshowninFigureS2. Of the three different intragenic mutations in OTX2, two
were plausible loss-of-function mutations, both deletions
resulting in a frameshift within the ORF. The remaining
Ocular phenotype
change is a missense mutation with the potential to alter
Each of the 10 SOX2 intragenic mutation cases reported splicing,p.(Gln83His).ResidueGln83lieswithinthehome-
here is associated with severe ocular malformation odomain of OTX2 between the second and third alpha
affecting at least one eye (Fig. 2). BA or UA with non- helical regions. This residue is conserved in all available
functioning microphthalmia (UA-UM) was present in arthropodorthologs,theorthodenticleprotein(Table 4).
four and two children, respectively. One of the children
with UA-UM (CaseID 3303) did fixate light and objects
Ocular phenotype
as a small child but lost her vision due to sclerocornea
and severe microcornea. Two families with transmission Three of the four individuals with intragenic mutations of
of a mutation from an affected mother to an affected OTX2 had severe malformations affecting at least one eye
child (FamID 3432 and 3227) were identified. In FamID with two affected individuals showing complete blindness
3432, the mother and son both had UA, on the left and (Fig. 4). One of the affected children showed BA (CaseID
right side, respectively. No ocular anomalies were seen in 2896). CaseID 2867 has right anophthalmia and microph-
the other eye. In FamID 3227, the mother had bilateral thalmia, microcornea and retinal coloboma in the left eye.
iris and chorio-retinal coloboma with microcornea in the The parents had observed some visual interaction in the
more affected left eye. Her first daughter showed a nor- first months of life, however, these were lost because of
mal eye examination. Her second daughter was born with the severe ocular developmental anomalies. Her mother
right anophthalmia and microphthalmia with microcror- (CaseID 3362), who carries the same mutation, demon-
nea and chorio-retinal coloboma without fixation. strated a reduced vision due to amblyopia in the left eye
but no coloboma or axial length abnormalities. Unfortu-
nately,shedidnotconsentforfurtherinvestigationsofret-
Extraocular phenotype and developmental data
inal function (electroretinogram) or morphology (optical
The mean Z-scores for birth weight and birth occi- coherence tomography). CaseID 3197 had anophthalmia
pitofrontal circumference (OFC) were 0.12 and −1.03, intherightbutnoocularabnormalitiesinthelefteye.
respectively. None of the birth weights were >2 SD from
the population mean. Two affected individuals (CaseID
Extraocular phenotype and developmental data
2813 and 3303) had congenital microcephaly (birth OFC
Z-score (cid:1)(cid:3)2 SD). Postnatal head growth was signifi- Therewassignificantpostnatalfailureofheadgrowth.The
cantly impaired with 6/8 cases having significant micro- mean Z-scores for birth weight and birth OFC were −0.56
cephaly. The mean Z-scores for postnatal OFC, height, and 0.02. The mean Z-scores for postnatal OFC, height,
ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc. 19
GeneticCausesofAnophthalmia C.Gerth-Kahlertetal.
Table2. PhenotypeinpatientswithmutationinSOX2gene.
3432 3194 2813 3171
FamID
CaseID 3432 3433 3194 2813 3171
Nucleotidechange c.70_89del20 c.70_89del20 c.138_140dupTGC c.244_245delTT c.302A>G
Predicted p.(Asn24Argfs*65) p.(Ala47dup) p.(Leu82Valfs*13) p.(His101Arg)
protein
change
orgenomic
coordinates
Paternal/maternal ?/het(affected) wt/wt wt/wt ?(unavailable)/wt ?(unavailable)/wt
genotype
Ancestral German German German German Croatian
background
Sex Male Female Male Male Male
Gestationalage 37 41 42 38+6
(weeks+days)
Birthweight(g) 3700[0.3] 3830[0.91] 3440[−0.32] 2950[−1.27]
[Z-score]
Birth 34.5[−0.55] 33.7[−1.18] 31.5[−2.9]
occipitofrontal
circumference
(cm)[Z-score]
Height(cm)[Z-score] 101[−0.36] 178[2.35] 110[−1.2] 80[−0.62] 70[1.93]
Weight(kg)[Z-score] 18 57[−1.97] 19[−0.72] 10[−1.27] 8.8[1.43]
Ageatgrowth 4years 28.5years 6years 1.5years 5months
measurement
Occipitofrontal 51at4[−0.083] 45.5at4[−4.5] 47.8at3.8[−2.9]
circumference
(cm)atage
(years)[Z-score]
Ageatlast 3.0years 27.5years 2years 1.5years 5months
assessment
Ocularphenotype RE LE RE LE RE LE RE LE RE LE
Clinical x x x x x x x x
anophthalmia
Microphthalmia
(axiallength,mm)
Microcornea
(diameter,mm)
Sclerocornea
Coloboma
Noabnormalities x x
Vision(decimal) No Fixation 0.7 No No No No No No No
Extraocular
abnormalities
CerebralMRI Cavumvergae Notdone Smallposterior Fronto-temporal Smallseptum
(atage) (2.9years) corpuscallosum, cerebralvolume pellucidumcyst
septum reduction (2months)
pellucidumcyst (3months)
(3months)
Hearing Normal?(OAEs Notest OAEsat6years OAEsatbirthand Notestavailable
atage3negative available normal age4years
buthadEustachian normal
catarrh)
Speech Delayed Reduced Good Severedelayed
development (babybabble)
Motor Delayed Spasticgait Walkedat2years Walkswith
development support
Sleep Normal Normal Normal Wakesupatnight
irregularly
ThegenomicsequenceidentifiersforSOX2areNT_005612.15GI:88966845.het,heterozygous;LE,lefteye;MRI,magneticresonanceimaging;
OAEs,otoacusticemissions;RE,righteye;wt,wild-type;?,unknown.
20 ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc.
C.Gerth-Kahlertetal. GeneticCausesofAnophthalmia
Table2.(continued).
3227 3303 3370 2850 3797
3227 3228 3303 3370 2850 3797
c.368A>G c.368A>G c.479_480 c.841_851 1.6Mbdel c277G>T
dupAC del11insA
p.(Asp123Gly) p.(Ala161 p.(Ala281 chr3: p.(Glu93*)
Thrfs*4) Argfs*87) 182,649,000
–
184,339,000
(hg18)
wt/het(affected) wt/wt wt/wt ?(unavail- wt/wt ?/wt
able)/wt
Austrian Austrian German German Kirgigistan/ German
Sachalin
Female Female Female Male Female Female
40 40 38 32+5 40+4 39+4
3250[−0.34] 3050[−0.79] 3090[−0.7] 2000[−0.18] 3840[0.94] 4210[1.7]
34[−0.44] 32[−2.06] 34[−0.44] 35[0.37]
85[0.28] 167[0.53] 110[−2.1] 80[−4.7] 120[−1.8] 78[−0.91]
10.9[−0.58] 59[−1.64] 20[−0.96] 10[−4.04] 23[−1.1] 10[−0.75]
1.9years 34years 7years 3.4years 8.5years 1.5years
48at3[−1.9] 45.6at2 46.0at5 49at8.9 44at1.5[−3.36]
[−2.8] [−4.4] [−3.4]
1.7years 34years 6years 2.4years 8.5years 3months
RE LE RE LE RE LE RE LE RE LE RE LE
x x x x x x x
x(18.5) x x(16.3mm)
x(8.5) x x x(497)
x x
Retinal/ Iris/retinal/ Iris/retinal/
choroidal choroidal choroidal
No No 1.0 0.025 No No No No No No No LP
fixation
Normal Notdone Frontal Notdone Pinealcyst Normal(1month)
(10days) cerebral (4.5years)
volume
reduction
(5.8years)
Normal Normal OAEsafter OAEsatbirth OAEsatbirth Normal
birth normal normal
abnormal
Normal Normal Severe Severe Normal Normal
delayed delayed
(only (baby
mommy, babble)
daddy)
Delayed Normal Severe Severe Normal Normal
delayed/ delayed/
cannotsit cannotsit
orwalk orwalk
Normal Normal Normal Normal Normal Normal
ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc. 21
GeneticCausesofAnophthalmia C.Gerth-Kahlertetal.
Table3. PhenotypeinpatientswithOTX2mutation.
2896 3197 2867
FamID
CaseID 2896 3197 2867
Nucleotidechange c.234delC c.249G>T c.276_294del19
Predicted p.(Glu79Serfs*30) p.(Gln83His)orp.? p.(Lys92Asnfs*11)
proteinchange
Paternal/maternalgenotype ?/wt wt/? wt/het(unaffected)
Ancestralbackground German ArabicEmirates German
Sex Male Female Female
Gestationalage 41+5 36
(weeks+days)
Birthweight(g)[Z-score] 3870[0.31] 2400[−0.55]
Birthoccipitofrontal 34[−0.95] 33[−1.2]
circumference
(cm)[Z-score]
Height(cm)[Z-score] 98[−3.6] 142[2.19] 113[0.93]
Weight(kg)[Z-score] 13[−4.3] 39[1.8] 13[−2.8]
Ageatgrowth 6 8.6 5
measurement(years)
Occipitofrontal 48.9at5.7[−2.7] Nomicrocephaly 47.7at5[−3.3]
circumference(cm)
atage(years)[Z-score]
Ageatlast 6 8.6 6
assessment(years)
Ocularphenotype RE LE RE LE RE LE
Clinicalanophthalmia x x x x
Microphthalmia x(15.0)
(axiallength,mm)
Microcornea x(8.0)
(diameter,mm)
Sclerocornea
Coloboma Retinal
Other Noirisvisible,
dysplastic
retina
Noabnormalities x
Vision(decimal) No No No 1.0 No No
Extraocularabnormalities
CerebralMRI(atage) Notractusopticus Normal(6months) Normal(3months)
(3months)
Hearing RightOAEatbirth Normal Normal
normal
Extremities Talipesequinovarus
Earmalformation LeftmicrotiaII°
Speechdevelopment Severedelayed Normal Delayed
(babybabble)
Motordevelopment Severedelayed, Normal Delayed(walksshortdistances
doesnotcrawl/ w/osupport
walk
Sleep Sleepdisorders Normal Sleepdisorders(abnormalSDSC)
(abnormalSDSC)
ThegenomicsequenceidentifiersforOTX2areNT_026437.11GI:51493278.ABR,auditorybrainstemresponse;BERA,brainstemevokedresponse
audiometry;LE,lefteye;MRI,magneticresonanceimaging;OAEs,otoacusticemissions;RE,righteye;SDSC,sleepdisturbancescaleforchildren.
22 ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc.
C.Gerth-Kahlertetal. GeneticCausesofAnophthalmia
Table3.(continued).
3346 3000
3362 3346 3000
c.276_294del19 455kbdeletion 6.5Mbdeletion
chr14:56,224,000- chr14:56,094,000-
56,679,000(hg18) 62,594,000(hg18)
?/? wt/wt ?/?
German Polish German
Female Male Female
39+2 39
2920[−0.98] 2810[−1.01]
38[2.2]
95[0.9] 112[−1.7]
12.5[−0.88] 19[−1.3]
2.6 7
49at3[−1.7] 47at8.8[−5.1]
34 1 8.7
RE LE RE LE RE LE
x x x x
Amblyopia
x
1.0 0.2 No No No No
Notdone Normal(1day) Smallpituitarygland(4.7years)
ABRandOAEsat8monthsnormal BERAatage
2.5years:good
responsesfrom
60to30db
Normal Normal Delayed
Normal Normal Delayed
Normal Normal Wakesupatnight
ª2013TheAuthors.MolecularGenetics&GenomicMedicinepublishedbyWileyPeriodicals,Inc. 23