doi: 10.1111/ahg.12090
Identification of Six Novel Mutations in ZEB1 and Description of the Associated Phenotypes in Patients with Posterior Polymorphous Corneal Dystrophy 3 Cerys J. Evans1,† , Petra Liskova1,2,3∗,† , Lubica Dudakova2 , Pavlina Hrabcikova4 , Ales Horinek5,6 , Katerina Jirsova2 , Martin Filipec7 , Alison J. Hardcastle1 , Alice E. Davidson1 and Stephen J. Tuft1,8 1 UCL Institute of Ophthalmology, London, UK 2 Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 3 Department of Ophthalmology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 4 Department of Ophthalmology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic 5 3rd Department of Medicine—Department of Endocrinology and Metabolism, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 6 Institute of Biology and Human Genetics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 7 Lexum European Eye Clinic, Prague, Czech Republic 8 Moorfields Eye Hospital NHS Foundation Trust, London, UK
Summary Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584∗ ) and c.1717_1718del; p.(Val573Phefs∗ 12) in two Czech families, c.1176dup; p.(Ala393Serfs∗ 19), c.1100C>A; p.(Ser367∗ ), c.627del; p.(Phe209Leufs∗ 11) in three British families and a splice site mutation, c.685–2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs∗ 3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5–52.7 diopters, steep keratometry 48.1–54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation. Keywords: ZEB1, posterior polymorphous corneal dystrophy type 3, phenotype
Introduction ∗
Corresponding author: PETRA LISKOVA, Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General University Hospital in Prague and First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, 128 08, Prague, Czech Republic. Tel: +420 224 967 139; E-mail: [email protected] † The first two authors contributed equally to the study.
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Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant disorder. Corneal changes include islands of abnormal endothelial cells with vesicular lesions and thickening of Descemet membrane (Krachmer, 1985). There may also be iridocorneal adhesions, pupillary uveal ectropion, and secondary glaucoma from growth of abnormal corneal endothelium over the trabecular meshwork (Rodrigues et al., 1980; Krachmer, 1985).
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PPCD is genetically heterogeneous. PPCD1 (OMIM 122000) has been associated with mutations in VSX1 (Heon et al., 2002) although in some families linkage and haplotype analysis suggests that the disease is caused by changes in an as yet undiscovered adjacent gene (Gwilliam et al., 2005; Liskova et al., 2012). PPCD2 (OMIM 609140) is associated with mutations in COL8A2 (Biswas et al., 2001), and PPCD3 (OMIM 609141) is caused by mutations in the transcription factor ZEB1 (Krafchak et al., 2005). It has been estimated that pathogenic variants in ZEB1 cause approximately one third of PPCD cases, although frequencies differ between the reported patient cohorts (Krafchak et al., 2005; Aldave et al., 2007; Vincent et al., 2009; Lechner et al., 2013; Liskova et al., 2013; Bakhtiari et al., 2013). Patients with PPCD3 also have a higher rate of abdominal hernia compared to the normal population (Krafchak et al., 2005). In one study, maldevelopment of the corpus callosum was noted in two PPCD3 probands (Jang et al., 2014). In this study, we identified ZEB1 disease-causing mutations in two Caucasian Czech, four white British, one Sri Lankan family affected with PPCD3, and report the associated phenotypes.
97.5 percentile (Gilani et al., 2013). One patient was examined using noncontact specular microscopy (Topcon SP2000P, Tokyo, Japan).
Amplification and Sanger Sequencing of the ZEB1 Gene DNA for affected probands, and any additional available family members, was extracted from venous blood (Gentra Puregene, Qiagen, Hilden, Germany) or saliva (Oragene, DNA Genotek, Inc., Kanata, Canada). PCR primers for amplification and sequencing of the ZEB1 gene were designed to cover the entire coding sequence and flanking intronic sequences (NCBI Reference Sequence NM_030751.5; Supplementary Material; Table S1). Bi-directional Sanger sequencing was performed as previously described (Liskova et al., 2007), and samples were analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). DNASTAR package software version 10.1.2 (Lasergene, Madison, WI, USA) was used for analysis of sequences compared to the reference sequence. We also performed targeted screening for the presence of mutations identified in any available family members.
Materials and Methods The study was approved by the relevant local research ethics committees and adhered to the tenets set out in the Helsinki Declaration. All participants signed informed consent before entering the study.
Clinical Assessment We examined eight Caucasian Czech probands from families C16, C24-C30, four Caucasian British probands from families B3, B5-B7, and one Sri Lankan proband with PPCD, and their available family members. We recorded the bestcorrected visual acuity (BCVA) using the Snellen annotation but converted to decimal values. Intraocular pressure (IOP) was measured by Goldmann applanation tonometry. Corneal topography was performed and the simulated keratometry values recorded by Pentacam (Oculus Optikger¨ate GmbH, Wetzlar, Germany), in two patients Topcon OM-4 Ophthalmometer (Topcon Medical Systems, Inc., Oakland, NJ, USA) was used. The following values were taken as normal: flat keratometry (K1) in the 3 mm central corneal zone 46.1 D, steep keratometry (K2) in the 3 mm central corneal zone 47.4 D, corneal thickness 601 μm at the thinnest point. These values are based on Pentacam measurements performed in 341 predominantly Caucasian adults and represent
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Analysis of Identified ZEB1 Variants Sequence variants were described according to the Human Genome Variation Society guidelines (http://www.hgvs. org/mutnomen/), and with reference to the NCBI Reference Sequences NM_030751.5 and NG_017048.1. Variants were also analyzed using Mutalyzer 2.0.beta-26 (https: //mutalyzer.nl/; Wildeman et al., 2008) and checked for population frequencies in the 1000 Genomes Project (http:// browser.1000genomes.org/index.html) and the Exome Variant Server (NHLBI Exome Sequencing Project; http://evs. gs.washington.edu/EVS/; both accessed June 10, 2014). To predict whether the c.685–2A>G variant affects pre-mRNA splicing of the ZEB1 transcript, the wild-type and mutated sequences of exon 6 and the surrounding intronic regions were analyzed using the splice site prediction tools Human Splicing Finder (HSF; http://www.umd.be/HSF/; Desmet et al., 2009), NNSPLICE (http://www.fruitfly.org/seq tools/ splice.html) (Reese et al., 1997), and NetGene2 (http:// www.cbs.dtu.dk/services/NetGene2/) (Brunak et al., 1991). Sequence data for British and Czech probands who were previously reported to be negative for mutations in VSX1, COL8A2, and ZEB1 (families C15-C17, B3 and B4) was also reanalyzed as described above (Liskova et al., 2007).
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Figure 1 Posterior polymorphous corneal dystrophy 3 families and sequence chromatograms of disease-associated ZEB1 mutations. (A) Czech family C16 with a c.1749_1750del; p.(Pro584∗ ) mutation. (B) Czech family C24 harboring a c.1717_1718del; p.(Val573Phefs∗ 12) mutation. (C) British family B3 with a c.1176dup; p.(Ala393Serfs∗ 19) mutation. (D) British family B5 harboring a c.1100C>A; p.(Ser367∗ ) mutation. (E) British family B6 with a c.1576dup; p.(Val526Glyfs∗ 3). (F) British family B7 with a c.627del; p.(Phe209Leufs∗ 11) mutation. (G) Sri Lankan family S1 harboring a splice site mutation c.685–2A>G. In individuals available for testing -/+ indicates the presence of the identified mutation in a heterozygous state, -/- represents wild type. Subjects clinically examined by one of the authors or those with available medical records are denoted with ∗ . Individuals who were not clinically and genetically evaluated and had no history of corneal disease may be asymptomatic carriers. C
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Testing for Zygosity The zygosity of a twin pair (pedigree C24; Fig. 1B) was established using the PowerPlex 16 HS System (Promega Corporation, Madison, WI, USA) which co-amplifies the following loci: D18S51, D21S11, TH01, D3S1358, Penta E, FGA, TPOX, D8S1179, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818, and Penta D (Ensenberger et al., 2010).
Results ZEB1 Mutations Causing PPCD3 Six novel and one previously reported ZEB1 mutations were identified in the heterozygous state in seven probands with PPCD3 (Fig. 1 and Table 1). Four of the changes identified are frameshift mutations predicted to lead to a premature termination codon, and two variants are nonsense mutations. The c.685–2A>G variant detected in family S1 is located at the highly conserved canonical splice acceptor site of exon 6, and is therefore predicted to cause aberrant pre-mRNA splicing of the ZEB1 transcript in vivo. Patient derived RNA was not available for analysis; therefore we tested this hypothesis using in silico splice prediction tools. All three algorithms used predicted the presence of the known splice acceptor site in the wild-type sequence (Table 3). The presence of the c.685–2A>G mutation led to the prediction that the splice acceptor site would be abolished. This may lead to exon 6 skipping, resulting in the production of a transcript containing a frameshift and a premature termination codon p.(Arg229Glufs∗ 24). Alternatively, a cryptic splice acceptor site may be activated in the absence of the normal splice acceptor site. All three programs that were used identified the most likely alternative splice acceptor site to be situated 440 bp upstream of exon 6 (Table 2). If this scenario was to occur in vivo the resulting transcript would encode a truncated protein product; p.(Arg229Aspfs∗ 9). None of the presumed pathogenic heterozygous mutations were found in the population databases checked (1000 Genomes Project and the Exome Variant Server). In addition, the mutations segregated with the disease phenotype in all families where additional members were available for molecular genetic testing (Fig. 1). Dizygosity was confirmed for twin brothers (individuals II:2 and II:3) from family C24 (Fig. 1B) by genotyping forensic markers (data not shown).
Phenotype of PPCD3 Families with ZEB1 Mutations The clinical characteristics of the study participants affected with PPCD3 are summarized in Table 3. In all cases, their
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affected status was confirmed by the presence of the diseasecausing mutation. Proband III:1 from Czech family C16 (Fig. 1A) was noted to have corneal opacity at birth that had partially resolved by the age of 7 months. Glasses were prescribed at 10 months of age and divergent strabismus was noted at 12 months. When he was last examined at the age of 22 years both corneas were diffusely opaque and there was a mild rotatory nystagmus. The patient had surgical correction of cryptorchidism at the age of 3 years. His mother (II:1) was diagnosed with bilateral glaucoma at the age of 42 years and although there are bilateral vesicular lesions and mild irregular opacification at the level of the Descemet membrane layer she is asymptomatic. She has vertebral bone spurs. Both II:1 and III:1 denied the presence of other extraocular features reported to be associated with PPCD3 (Krafchak et al., 2005). The grandmother (I:2) underwent bilateral penetrating keratoplasty (PK) and had glaucoma but no other details are available. Individual I:1 from Czech family C24 (Fig. 1B) had band keratopathy for which he had at least one corneal debridement. He was registered as blind in his fourth decade but declined treatment and was lost to follow up. His parents were clinically normal. Siblings (II:1, II:2, and II:3) had worn glasses since the age of 6 years and had characteristic endothelial changes consistent with the diagnosis of PPCD3 (Fig. 2C). Twin II:2 also had bilateral iris flocculi (Fig. 2A, B). Both twins II:2 and II:3 suffered from congenital umbilical hernia that resolved spontaneously. Proband II:1 from British family B3 (Fig. 1C) developed bilateral corneal edema at the age of 18 months. A left PK was performed at 3 years of age followed by a right PK at 9 years of age. He has now had a total of three right PKs and seven left PKs. He did not report any extraocular features. His father (I:1) has a milder phenotype with normal vision without surgery, although a detailed ocular examination was not available. The father had two operations for umbilical hernia. The proband II:1 from family B5 (Fig. 1D) had poor vision since childhood and had surgery for strabismus at the age of 7 years. Apart from changes in the posterior corneal layers there were no other documented ocular abnormalities. The father (I:1) of the proband was diagnosed with PPCD at the age of 4 years, and although his parents were not examined, both were known to have good visual acuity. No systemic abnormalities were documented in I:1 and II:1, but specific enquiry about the extraocular features associated with PPCD3 was not possible as the family was lost to follow-up. The proband from family B6 (Fig. 1E) was first seen at the age of 32 years when she was referred for a PK in the right eye. She subsequently had a further three grafts in the same eye. There was no history of ocular disease in her parents.
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Table 1 ZEB1 disease-causing mutations identified in probands with posterior polymorphous corneal dystrophy 3. Family ID
Origin
Mutation type
Mutation description
Predicted effect on protein
C16 C24 B3 B5 B6 B7 S1
Caucasian Czech Caucasian Czech Caucasian British Caucasian British Caucasian British Caucasian British Sri Lankan
Nonsense Frameshift Frameshift Nonsense Frameshift Frameshift Splicing
c.1749_1750del c.1717_1718del c.1176dup c.1100C>A c.1576dup c.627del c.685–2A>G
p.(Pro584∗ ) p.(Val573Phefs∗ 12) p.(Ala393Serfs∗ 19) p.(Ser367∗ ) p.(Val526Glyfs∗ 3) p.(Phe209Leufs∗ 11) Unknown
Human Genome Variation Society nomenclature was used to describe sequence variants for transcript NM_030751.5. The mutation identified in family B6 has been previously reported in three other probands of US origin (Krafchak et al., 2005; Lechner et al., 2013; Bakhtiari et al., 2013). Table 2 In silico analysis of the c.685–2A>G ZEB1 variant identified in the proband from family S1. Splice prediction tool
Wild-type splice acceptor site
Splice acceptor site with the c.685– 2A>G mutation
Alternative cryptic splice acceptor site 440 bp upstream in presence of c.685–2A>G
Human Splice Finder (0–100) NNSPLICE (0–1) NetGene2 (0–1)
93.2 0.98 0.85
0 0 0
92.89 0.90 0.16
Potential splice acceptor sites in a given sequence are scored by Human Splicing Finder, NNSPLICE and NetGene2 from 0 to 100, 0—1, and 0–1, respectively. In each instance, a higher number corresponds to an increased likelihood of the motif functioning as a splice acceptor site. An alternative splice acceptor site is predicted by all three in silico programs 440 bp upstream of ZEB1 exon 6 (NM_030751.5 and NG_017048.1).
Figure 2 Anterior segment findings in patients with posterior polymorphous corneal dystrophy 3. (A) Iris flocculi in the left eye of individual III:2 from family C24, (B) further documented in an enhanced Scheimpflug image (arrow); imbedded image shows axis of the image capture. (C) Typical vesicular lesions with gray halos in the right eye of individual III:3 from family C24 (D) and in the right eye of individual II:1 from family S1 in retroillumination. (E) Specular microscopy imaging of the right eye of individual II:1 from family S1 showing irregular posterior corneal surface and abnormalities of corneal endothelial cell morphology.
Details of systemic features were unavailable as the patient was lost to follow up. Individual II:1 from family B7 (Fig. 1F) reported poor vision since childhood which gradually deteriorated. He was first examined at Moorfields Eye Hospital at the age of 38 years. At the age of 39 years he underwent left PK followed
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by a right PK when he was 41 years old. Except for corneal changes consistent with the diagnosis of PPCD no other abnormalities of the anterior segment were noted. The patient reported poor vision in his father, however, no further details could be obtained. His three siblings had no eyesight problems but were not available for examination. The proband
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I:1/M II:2/M I:1/F II:1/M II:1/M
B5
39 19 32 38 29
44 22 16 10 10 37
Age (yrs)
0.33 0.25 0.1 0.25 0.78
0.66 0.25 0.66 0.5 0.66 CF
RE
0.33 0.33 0.33 0.1 0.6
0.66 0.16 0.66 0.25 0.5 0.66
LE
NA NA NA NA 608
599 601 628 610 666 NA
RE
NA NA NA NA 597
591 609 634 606 657 NA
LE
Thinnest point
Y N N N N Steroid responder N N N N N
Glaucoma
NA −7.00 −1.50 × 10° NA +1.00 NA
NA −8.00 −2.25 × 180° −3.75 −0.25 × 110° −1.75 −2.5 × 180° +0.25 −0.5 × 170° NA
RE
Refraction (DS/DC)
NA −6.00 −0.50 × 90° −1.5 −1.5 × 160° +2.00 NA
NA −3.0 −3.0 × 180° −4,25 −0.5 × 36° −2.75 −2.25 × 180° −0.5 −1.5 × 180° NA
LE
NA 52.7/53.6 PK 46.5/46.9 45.9/48.1
45.2/46.4 50.4/50.8 44.4/44.8 49.5/53.4 44.2/45.7 PK
RE
K1/K2 (D)∗
NA 52.7/52.7 NA 43.5/44.2 47.0/48.4
45.5/46.5 47.7/48.6 45.0/46.9 50.5/54.0 46.0/48.1 PK
LE
BCVA, best corrected visual acuity; CF, counting fingers; D, diopter; DS, diopter sphere; DC, diopter cylinder; F, female; K1, corneal dioptric power in the flattest meridian for the 3 mm central zone; K2, corneal dioptric power in the steepest meridian for the 3 mm central zone; LE, left eye; RE, right eye; M, male; N, no; NA, not available; PK, penetrating keratoplasty; Y, yes;. Yrs, years. Simulated K values and thinnest point were measured in individuals from families C16, C24 and S1 using Pentacam. Topcon OM-4 was used for measurements of K values in subject II:2 from family B4 and probands from families B5 and B6. Abnormal values > 97.5 percentile (>46.1 D for K1, >47.4 D for K2, and >601 μm for thinnest pachymetry [Gilani et al., 2013]) are indicated in bold.
B6 B7 S1
B3
C24
III:1/F IV:1/M II:1/M II:2/M II:3/M II:1/M
Indivi-dual /Gender
C16
Family
BCVA
Table 3 Clinical characteristics of 11 cases with posterior polymorphous corneal dystrophy 3.
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had a history of an enlarged heart since childhood and was indicated for pericardiectomy when he was 37 years old. The proband II:1 from family S1 (Fig. 1F) of Sri Lankan origin had a negative family history for corneal disease although no other family members were available for examination. He was noted to have poor vision at 7 years of age. At the age of 29 years he was suspected to have glaucoma, with IOP measurements of 24 mmHg in the left eye, but with normal optic discs and open anterior chamber angles. Vesicular endothelial lesions (Fig. 2D) and specular microscopy showed enlarged endothelial cells and uneven posterior corneal surface (Fig. 2E). He had a right posterior lamellar keratoplasty with a subsequent BCVA of 0.18 despite a clear graft, suggesting the presence of amblyopia. The patient did not report any general health problems however precise details on extraocular features associated with PPCD3 were not available. The majority (10 out of 16) of patient corneas had steep keratometry values. Pachymetry was increased in 8 out of 12 eyes (Table 3).
Discussion In this study, we report six novel ZEB1 mutations causing PPCD3. We confirm that the disease may present as corneal edema detected at, or soon after, birth (Liskova et al., 2010). In addition, consistent with previous reports, the majority of corneas had an abnormally steep curvature and thickness (Liskova et al., 2010; Raber et al., 2011; Liskova et al., 2013; Aldave et al., 2013). We were also able to compare the phenotype in a pair of male dizygotic twins, who had differences in the degree of corneal steepening and the presence of iris flocculi. To the best of our knowledge this is the first time iris flocculi have been observed in a patient with PPCD3. We suggest that cryptorchidism may also be a part of the PPCD3 phenotype. The prevalence of cryptorchidism in the general population is 1.2% at 1 month of age (Pierik et al., 2005) and 1.1% at 1 year (Berkowitz et al., 1993), while an undescended testicle has to date (including this study) been reported in three of the 50 males with PPCD3 (Krafchak et al., 2005; Aldave et al., 2007; Liskova et al., 2007; Nguyen et al., 2010; Vincent et al., 2009; Lechner et al., 2013; Liskova et al., 2013; Bakhtiari et al., 2013). PPCD has been reported to be caused by mutations in at least three genes (Biswas et al., 2001; Heon et al., 2002; Gwilliam et al., 2005; Krafchak et al., 2005). To date, the majority of Caucasian British PPCD probands (five of seven, 71%) attending Moorfields Eye Hospital have been shown to harbor a causative mutation in ZEB1 coding exons (current study and Liskova et al., 2007). This is in contrast to the reports of US cohorts identifying ZEB1 mutations in approximately 30% of cases (Krafchak et al., 2005; Aldave et al., 2007;
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Bakhtiari et al., 2013), highlighting differences between patient populations. In the Czech Republic, six out of 30 (20%) of probands have a causative mutation in ZEB1 coding exons. This relatively low frequency is likely influenced by the fact that over 50% of Czech PPCD probands are presumed to be descendants of a common founder harboring a mutation within the PPCD1 locus (Liskova et al., 2012). Four of the five novel ZEB1 mutations we identified are predicted to produce a transcript containing a premature termination codon that will be degraded by nonsense mediated decay (NMD) or produce a truncated protein resulting in loss of functional domains, which is consistent with previous studies (Krafchak et al., 2005; Aldave et al., 2007; Liskova et al., 2007; Nguyen et al., 2010; Lechner et al., 2013). It has been estimated that at least 10% of all mutations causing human inherited disease affect pre-mRNA splicing (Krawczak et al., 2007). RNA from the individual with a c.685–2A>G variant was not available for further analysis; therefore, we used in silico splice prediction algorithms to assess its likely effect. Results of the analysis predicted that the mutation would cause aberrant pre-mRNA splicing of the ZEB1 transcript in vivo, and that this would lead to premature termination and either NMD or a truncated protein product. All ZEB1 mutations identified to date are unique to the families, with the exception of the c.1576dup mutation, which has been now observed for the fourth time (Krafchak et al., 2005; Bakhtiari et al., 2013; Lechner et al., 2013), suggesting this may be a mutation hotspot. As a cautionary note, probands from families C16 and B3 were previously reported as ZEB1 negative (Liskova et al., 2007). Reanalysis of the sequence data indicated that their disease-causing changes had been missed in this previous study due to their localization at the end of a PCR fragment, leading to seemingly wild-type sequence assembly. In this study individual I:1 from families C24, B3, B5, B6, and probands from families S1 and B7 did not have a family history of the disease. This may be due to asymptomatic status in one of the parents, disease nonpenetrance or due to the mutation(s) arising de novo. Nonpenetrance and a de novo origin of mutations have been previously documented for PPCD3 (Krafchak et al., 2005; Liskova et al., 2007; Liskova et al., 2010; Bakhtiari et al., 2013). However, because of lack of additional familial samples further molecular genetic investigation could not be performed. Nonocular phenotypes, associated with ZEB1 mutations have been reported previously, most commonly the presence of hernia (Krafchak et al., 2005; Aldave et al., 2007; Lechner et al., 2013; Liskova et al., 2013; Jang et al., 2014). In this study, there was a self-reported history of umbilical hernia in the twins from family C24, and umbilical hernia in the father of the proband from family B3. The possible association of PPCD3 with cryptorchidism warrants further investigation.
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Acknowledgements This work was supported by research grant GACR P301/12/P591 (PL). Institutional support was provided by the PRVOUK-P24/LF1/3 program of the Charles University in Prague. AJH and SJT are supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. CJE is supported by a Fight for Sight PhD studentship (AJH and SJT). AED is supported by the Lanvern Foundation, Moorfields Eye Charity and Moorfields Special Trustees (AJH and SJT), and the Rosetrees Trust (AED and AJH). LD was supported by the grant SVV-2014–260022.
Conflict of Interest The authors declare no conflict of interest.
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Developmental validation of the PowerPlex 16 HS System: An improved 16-locus fluorescent STR multiplex. Forensic Sci Int Genet 4, 257–264. Gilani, F., Cortese, M., Ambrosio, R. R., Jr., Lopes, B., Ramos, I., Harvey, E. M. & Belin, M. W. (2013) Comprehensive anterior segment normal values generated by rotating Scheimpflug tomography. J Cataract Refract Surg 39, 1707–1712. Gwilliam, R., Liskova, P., Filipec, M., Kmoch, S., Jirsova, K., Huckle, E. J., Stables, C. L., Bhattacharya, S. S., Hardcastle, A. J., Deloukas, P. & Ebenezer, N. D. (2005) Posterior polymorphous corneal dystrophy in Czech families maps to chromosome 20 and excludes the VSX1 gene. Invest Ophthalmol Vis Sci 46, 4480– 4484. Heon, E., Greenberg, A., Kopp, K. K., Rootman, D., Vincent, A. L., Billingsley, G., Priston, M., Dorval, K. M., Chow, R. L., Mcinnes, R. R., Heathcote, G., Westall, C., Sutphin, J. E., Semina, E., Bremner, R. & Stone, E. M. (2002) VSX1: A gene for posterior polymorphous dystrophy and keratoconus. Hum Mol Genet 11, 1029–1036. Jang, M. S., Roldan, A. N., Frausto, R. F. & Aldave, A. J. (2014) Posterior polymorphous corneal dystrophy 3 is associated with agenesis and hypoplasia of the corpus callosum. Vision Res 100, 88–92. Krafchak, C. M., Pawar, H., Moroi, S. E., Sugar, A., Lichter, P. R., Mackey, D. A., Mian, S., Nairus, T., Elner, V., Schteingart, M. T., Downs, C. A., Kijek, T. G., Johnson, J. M., Trager, E. H., Rozsa, F. W., Mandal, M. N., Epstein, M. P., Vollrath, D., Ayyagari, R., Boehnke, M. & Richards, J. E. (2005) Mutations in TCF8 cause posterior polymorphous corneal dystrophy and ectopic expression of COL4A3 by corneal endothelial cells. Am J Hum Genet, 77, 694–708. Krachmer, J. H. (1985) Posterior polymorphous corneal dystrophy: A disease characterized by epithelial-like endothelial cells which influence management and prognosis. Trans Am Ophthalmol Soc 83, 413–475. Krawczak, M., Thomas, N. S., Hundrieser, B., Mort, M., Wittig, M., Hampe, J. & Cooper, D. N. (2007) Single base-pair substitutions in exon-intron junctions of human genes: Nature, distribution, and consequences for mRNA splicing. Hum Mutat 28, 150–158. Lechner, J., Dash, D. P., Muszynska, D., Hosseini, M., Segev, F., George, S., Frazer, D. G., Moore, J. E., Kaye, S. B., Young, T., Simpson, D. A., Churchill, A. J., Heon, E. & Willoughby, C. E. (2013) Mutational spectrum of the ZEB1 gene in corneal dystrophies supports a genotype-phenotype correlation. Invest Ophthalmol Vis Sci 54, 3215–3223. Liskova, P., Filipec, M., Merjava, S., Jirsova, K. & Tuft, S. J. (2010) Variable ocular phenotypes of posterior polymorphous corneal dystrophy caused by mutations in the ZEB1 gene. Ophthalmic Genet 31, 230–234. Liskova, P., Gwilliam, R., Filipec, M., Jirsova, K., Reinstein Merjava, S., Deloukas, P., Webb, T. R., Bhattacharya, S. S., Ebenezer, N. D., Morris, A. G. & Hardcastle, A. J. (2012) High prevalence of posterior polymorphous corneal dystrophy in the Czech Republic; linkage disequilibrium mapping and dating an ancestral mutation. PLoS One 7, e45495. Liskova, P., Palos, M., Hardcastle, A. J. & Vincent, A. L. (2013) Further genetic and clinical insights of posterior polymorphous corneal Dystrophy 3. JAMA Ophthalmol 10, 1296– 1303. Liskova, P., Tuft, S. J., Gwilliam, R., Ebenezer, N. D., Jirsova, K., Prescott, Q., Martincova, R., Pretorius, M., Sinclair, N.,
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Boase, D. L., Jeffrey, M. J., Deloukas, P., Hardcastle, A. J., Filipec, M. & Bhattacharya, S. S. (2007) Novel mutations in the ZEB1 gene identified in Czech and British patients with posterior polymorphous corneal dystrophy. Hum Mutat 28, 638. doi: 10.1002/humu.9495 Nguyen, D. Q., Hosseini, M., Billingsley, G., Heon, E. & Churchill, A. J. (2010) Clinical phenotype of posterior polymorphous corneal dystrophy in a family with a novel ZEB1 mutation. Acta Ophthalmol 88, 695–699. Pierik, F. H., Burdorf, A., De Muinck Keizer-Schrama, S. M., Wolffenbuttel, K. P., Nijman, J. M., Juttmann, R. E. & Weber, R. F. (2005) The cryptorchidism prevalence among infants in the general population of Rotterdam, the Netherlands. Int J Androl 28, 248–252. Raber, I. M., Fintelmann, R., Chhabra, S., Ribeiro, M. P., Eagle, R. C. Jr. & Orlin, S. E. (2011) Posterior polymorphous dystrophy associated with nonkeratoconic steep corneal curvatures. Cornea 10, 1120–1124. Reese, M. G., Eeckman, F. H., Kulp, D. & Haussler, D. (1997) Improved splice site detection in Genie. J Comput Biol 4, 311– 323. Rodrigues, M. M., Phelps, C. D., Krachmer, J. H., Cibis, G. W. & Weingeist, T. A. (1980) Glaucoma due to endothelialization of the anterior chamber angle. A comparison of posterior
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polymorphous dystrophy of the cornea and Chandler’s syndrome. Arch Ophthalmol 98, 688–696. Vincent, A. L., Niederer, R. L., Richards, A., Karolyi, B., Patel, D. V. & Mcghee, C. N. (2009) Phenotypic characterisation and ZEB1 mutational analysis in posterior polymorphous corneal dystrophy in a New Zealand population. Mol Vis 15, 2544–2553. Wildeman, M., Van Ophuizen, E., Den Dunnen, J. T. & Taschner, P. E. (2008) Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker. Hum Mutat 29, 6–13.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Primer sequences used for amplification and sequencing ZEB1. Received: 9 July 2014 Accepted: 30 September 2014
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Identification of Six Novel Mutations in ZEB1 and Description of the Associated Phenotypes in Patients with Posterior Polymorphous Corneal Dystrophy 3 Cerys J. Evans1,† , Petra Liskova1,2,3∗,† , Lubica Dudakova2 , Pavlina Hrabcikova4 , Ales Horinek5,6 , Katerina Jirsova2 , Martin Filipec7 , Alison J. Hardcastle1 , Alice E. Davidson1 and Stephen J. Tuft1,8 1 UCL Institute of Ophthalmology, London, UK 2 Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 3 Department of Ophthalmology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 4 Department of Ophthalmology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic 5 3rd Department of Medicine—Department of Endocrinology and Metabolism, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 6 Institute of Biology and Human Genetics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 7 Lexum European Eye Clinic, Prague, Czech Republic 8 Moorfields Eye Hospital NHS Foundation Trust, London, UK
Summary Posterior polymorphous corneal dystrophy 3 (PPCD3) is a rare autosomal dominant disorder caused by mutations in ZEB1. To date all identified disease-causing variants were unique to the studied families, except for c.1576dup. We have detected six novel ZEB1 mutations; c.1749_1750del; p.(Pro584∗ ) and c.1717_1718del; p.(Val573Phefs∗ 12) in two Czech families, c.1176dup; p.(Ala393Serfs∗ 19), c.1100C>A; p.(Ser367∗ ), c.627del; p.(Phe209Leufs∗ 11) in three British families and a splice site mutation, c.685–2A>G, in a patient of Sri Lankan origin. An additional British proband had the c.1576dup; p.(Val526Glyfs∗ 3) mutation previously reported in other populations. Clinical findings were variable and included bilateral congenital corneal opacity in one proband, development of opacity before the age of 2 years in another individual and bilateral iris flocculi in yet another subject. The majority of eyes examined by corneal topography (10 out of 16) had an abnormally steep cornea (flat keratometry 46.5–52.7 diopters, steep keratometry 48.1–54.0 diopters). One proband underwent surgery for cryptorchidism. Our study further demonstrates that PPCD3 can present as corneal edema in early childhood, and that an abnormally steep keratometry is a common feature of this condition. As cryptorchidism has been previously observed in two other PPCD3 cases, its association with the disease warrants further investigation. Keywords: ZEB1, posterior polymorphous corneal dystrophy type 3, phenotype
Introduction ∗
Corresponding author: PETRA LISKOVA, Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, General University Hospital in Prague and First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, 128 08, Prague, Czech Republic. Tel: +420 224 967 139; E-mail: [email protected] † The first two authors contributed equally to the study.
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Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant disorder. Corneal changes include islands of abnormal endothelial cells with vesicular lesions and thickening of Descemet membrane (Krachmer, 1985). There may also be iridocorneal adhesions, pupillary uveal ectropion, and secondary glaucoma from growth of abnormal corneal endothelium over the trabecular meshwork (Rodrigues et al., 1980; Krachmer, 1985).
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PPCD is genetically heterogeneous. PPCD1 (OMIM 122000) has been associated with mutations in VSX1 (Heon et al., 2002) although in some families linkage and haplotype analysis suggests that the disease is caused by changes in an as yet undiscovered adjacent gene (Gwilliam et al., 2005; Liskova et al., 2012). PPCD2 (OMIM 609140) is associated with mutations in COL8A2 (Biswas et al., 2001), and PPCD3 (OMIM 609141) is caused by mutations in the transcription factor ZEB1 (Krafchak et al., 2005). It has been estimated that pathogenic variants in ZEB1 cause approximately one third of PPCD cases, although frequencies differ between the reported patient cohorts (Krafchak et al., 2005; Aldave et al., 2007; Vincent et al., 2009; Lechner et al., 2013; Liskova et al., 2013; Bakhtiari et al., 2013). Patients with PPCD3 also have a higher rate of abdominal hernia compared to the normal population (Krafchak et al., 2005). In one study, maldevelopment of the corpus callosum was noted in two PPCD3 probands (Jang et al., 2014). In this study, we identified ZEB1 disease-causing mutations in two Caucasian Czech, four white British, one Sri Lankan family affected with PPCD3, and report the associated phenotypes.
97.5 percentile (Gilani et al., 2013). One patient was examined using noncontact specular microscopy (Topcon SP2000P, Tokyo, Japan).
Amplification and Sanger Sequencing of the ZEB1 Gene DNA for affected probands, and any additional available family members, was extracted from venous blood (Gentra Puregene, Qiagen, Hilden, Germany) or saliva (Oragene, DNA Genotek, Inc., Kanata, Canada). PCR primers for amplification and sequencing of the ZEB1 gene were designed to cover the entire coding sequence and flanking intronic sequences (NCBI Reference Sequence NM_030751.5; Supplementary Material; Table S1). Bi-directional Sanger sequencing was performed as previously described (Liskova et al., 2007), and samples were analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). DNASTAR package software version 10.1.2 (Lasergene, Madison, WI, USA) was used for analysis of sequences compared to the reference sequence. We also performed targeted screening for the presence of mutations identified in any available family members.
Materials and Methods The study was approved by the relevant local research ethics committees and adhered to the tenets set out in the Helsinki Declaration. All participants signed informed consent before entering the study.
Clinical Assessment We examined eight Caucasian Czech probands from families C16, C24-C30, four Caucasian British probands from families B3, B5-B7, and one Sri Lankan proband with PPCD, and their available family members. We recorded the bestcorrected visual acuity (BCVA) using the Snellen annotation but converted to decimal values. Intraocular pressure (IOP) was measured by Goldmann applanation tonometry. Corneal topography was performed and the simulated keratometry values recorded by Pentacam (Oculus Optikger¨ate GmbH, Wetzlar, Germany), in two patients Topcon OM-4 Ophthalmometer (Topcon Medical Systems, Inc., Oakland, NJ, USA) was used. The following values were taken as normal: flat keratometry (K1) in the 3 mm central corneal zone 46.1 D, steep keratometry (K2) in the 3 mm central corneal zone 47.4 D, corneal thickness 601 μm at the thinnest point. These values are based on Pentacam measurements performed in 341 predominantly Caucasian adults and represent
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Analysis of Identified ZEB1 Variants Sequence variants were described according to the Human Genome Variation Society guidelines (http://www.hgvs. org/mutnomen/), and with reference to the NCBI Reference Sequences NM_030751.5 and NG_017048.1. Variants were also analyzed using Mutalyzer 2.0.beta-26 (https: //mutalyzer.nl/; Wildeman et al., 2008) and checked for population frequencies in the 1000 Genomes Project (http:// browser.1000genomes.org/index.html) and the Exome Variant Server (NHLBI Exome Sequencing Project; http://evs. gs.washington.edu/EVS/; both accessed June 10, 2014). To predict whether the c.685–2A>G variant affects pre-mRNA splicing of the ZEB1 transcript, the wild-type and mutated sequences of exon 6 and the surrounding intronic regions were analyzed using the splice site prediction tools Human Splicing Finder (HSF; http://www.umd.be/HSF/; Desmet et al., 2009), NNSPLICE (http://www.fruitfly.org/seq tools/ splice.html) (Reese et al., 1997), and NetGene2 (http:// www.cbs.dtu.dk/services/NetGene2/) (Brunak et al., 1991). Sequence data for British and Czech probands who were previously reported to be negative for mutations in VSX1, COL8A2, and ZEB1 (families C15-C17, B3 and B4) was also reanalyzed as described above (Liskova et al., 2007).
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Figure 1 Posterior polymorphous corneal dystrophy 3 families and sequence chromatograms of disease-associated ZEB1 mutations. (A) Czech family C16 with a c.1749_1750del; p.(Pro584∗ ) mutation. (B) Czech family C24 harboring a c.1717_1718del; p.(Val573Phefs∗ 12) mutation. (C) British family B3 with a c.1176dup; p.(Ala393Serfs∗ 19) mutation. (D) British family B5 harboring a c.1100C>A; p.(Ser367∗ ) mutation. (E) British family B6 with a c.1576dup; p.(Val526Glyfs∗ 3). (F) British family B7 with a c.627del; p.(Phe209Leufs∗ 11) mutation. (G) Sri Lankan family S1 harboring a splice site mutation c.685–2A>G. In individuals available for testing -/+ indicates the presence of the identified mutation in a heterozygous state, -/- represents wild type. Subjects clinically examined by one of the authors or those with available medical records are denoted with ∗ . Individuals who were not clinically and genetically evaluated and had no history of corneal disease may be asymptomatic carriers. C
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Testing for Zygosity The zygosity of a twin pair (pedigree C24; Fig. 1B) was established using the PowerPlex 16 HS System (Promega Corporation, Madison, WI, USA) which co-amplifies the following loci: D18S51, D21S11, TH01, D3S1358, Penta E, FGA, TPOX, D8S1179, vWA, Amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818, and Penta D (Ensenberger et al., 2010).
Results ZEB1 Mutations Causing PPCD3 Six novel and one previously reported ZEB1 mutations were identified in the heterozygous state in seven probands with PPCD3 (Fig. 1 and Table 1). Four of the changes identified are frameshift mutations predicted to lead to a premature termination codon, and two variants are nonsense mutations. The c.685–2A>G variant detected in family S1 is located at the highly conserved canonical splice acceptor site of exon 6, and is therefore predicted to cause aberrant pre-mRNA splicing of the ZEB1 transcript in vivo. Patient derived RNA was not available for analysis; therefore we tested this hypothesis using in silico splice prediction tools. All three algorithms used predicted the presence of the known splice acceptor site in the wild-type sequence (Table 3). The presence of the c.685–2A>G mutation led to the prediction that the splice acceptor site would be abolished. This may lead to exon 6 skipping, resulting in the production of a transcript containing a frameshift and a premature termination codon p.(Arg229Glufs∗ 24). Alternatively, a cryptic splice acceptor site may be activated in the absence of the normal splice acceptor site. All three programs that were used identified the most likely alternative splice acceptor site to be situated 440 bp upstream of exon 6 (Table 2). If this scenario was to occur in vivo the resulting transcript would encode a truncated protein product; p.(Arg229Aspfs∗ 9). None of the presumed pathogenic heterozygous mutations were found in the population databases checked (1000 Genomes Project and the Exome Variant Server). In addition, the mutations segregated with the disease phenotype in all families where additional members were available for molecular genetic testing (Fig. 1). Dizygosity was confirmed for twin brothers (individuals II:2 and II:3) from family C24 (Fig. 1B) by genotyping forensic markers (data not shown).
Phenotype of PPCD3 Families with ZEB1 Mutations The clinical characteristics of the study participants affected with PPCD3 are summarized in Table 3. In all cases, their
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affected status was confirmed by the presence of the diseasecausing mutation. Proband III:1 from Czech family C16 (Fig. 1A) was noted to have corneal opacity at birth that had partially resolved by the age of 7 months. Glasses were prescribed at 10 months of age and divergent strabismus was noted at 12 months. When he was last examined at the age of 22 years both corneas were diffusely opaque and there was a mild rotatory nystagmus. The patient had surgical correction of cryptorchidism at the age of 3 years. His mother (II:1) was diagnosed with bilateral glaucoma at the age of 42 years and although there are bilateral vesicular lesions and mild irregular opacification at the level of the Descemet membrane layer she is asymptomatic. She has vertebral bone spurs. Both II:1 and III:1 denied the presence of other extraocular features reported to be associated with PPCD3 (Krafchak et al., 2005). The grandmother (I:2) underwent bilateral penetrating keratoplasty (PK) and had glaucoma but no other details are available. Individual I:1 from Czech family C24 (Fig. 1B) had band keratopathy for which he had at least one corneal debridement. He was registered as blind in his fourth decade but declined treatment and was lost to follow up. His parents were clinically normal. Siblings (II:1, II:2, and II:3) had worn glasses since the age of 6 years and had characteristic endothelial changes consistent with the diagnosis of PPCD3 (Fig. 2C). Twin II:2 also had bilateral iris flocculi (Fig. 2A, B). Both twins II:2 and II:3 suffered from congenital umbilical hernia that resolved spontaneously. Proband II:1 from British family B3 (Fig. 1C) developed bilateral corneal edema at the age of 18 months. A left PK was performed at 3 years of age followed by a right PK at 9 years of age. He has now had a total of three right PKs and seven left PKs. He did not report any extraocular features. His father (I:1) has a milder phenotype with normal vision without surgery, although a detailed ocular examination was not available. The father had two operations for umbilical hernia. The proband II:1 from family B5 (Fig. 1D) had poor vision since childhood and had surgery for strabismus at the age of 7 years. Apart from changes in the posterior corneal layers there were no other documented ocular abnormalities. The father (I:1) of the proband was diagnosed with PPCD at the age of 4 years, and although his parents were not examined, both were known to have good visual acuity. No systemic abnormalities were documented in I:1 and II:1, but specific enquiry about the extraocular features associated with PPCD3 was not possible as the family was lost to follow-up. The proband from family B6 (Fig. 1E) was first seen at the age of 32 years when she was referred for a PK in the right eye. She subsequently had a further three grafts in the same eye. There was no history of ocular disease in her parents.
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Table 1 ZEB1 disease-causing mutations identified in probands with posterior polymorphous corneal dystrophy 3. Family ID
Origin
Mutation type
Mutation description
Predicted effect on protein
C16 C24 B3 B5 B6 B7 S1
Caucasian Czech Caucasian Czech Caucasian British Caucasian British Caucasian British Caucasian British Sri Lankan
Nonsense Frameshift Frameshift Nonsense Frameshift Frameshift Splicing
c.1749_1750del c.1717_1718del c.1176dup c.1100C>A c.1576dup c.627del c.685–2A>G
p.(Pro584∗ ) p.(Val573Phefs∗ 12) p.(Ala393Serfs∗ 19) p.(Ser367∗ ) p.(Val526Glyfs∗ 3) p.(Phe209Leufs∗ 11) Unknown
Human Genome Variation Society nomenclature was used to describe sequence variants for transcript NM_030751.5. The mutation identified in family B6 has been previously reported in three other probands of US origin (Krafchak et al., 2005; Lechner et al., 2013; Bakhtiari et al., 2013). Table 2 In silico analysis of the c.685–2A>G ZEB1 variant identified in the proband from family S1. Splice prediction tool
Wild-type splice acceptor site
Splice acceptor site with the c.685– 2A>G mutation
Alternative cryptic splice acceptor site 440 bp upstream in presence of c.685–2A>G
Human Splice Finder (0–100) NNSPLICE (0–1) NetGene2 (0–1)
93.2 0.98 0.85
0 0 0
92.89 0.90 0.16
Potential splice acceptor sites in a given sequence are scored by Human Splicing Finder, NNSPLICE and NetGene2 from 0 to 100, 0—1, and 0–1, respectively. In each instance, a higher number corresponds to an increased likelihood of the motif functioning as a splice acceptor site. An alternative splice acceptor site is predicted by all three in silico programs 440 bp upstream of ZEB1 exon 6 (NM_030751.5 and NG_017048.1).
Figure 2 Anterior segment findings in patients with posterior polymorphous corneal dystrophy 3. (A) Iris flocculi in the left eye of individual III:2 from family C24, (B) further documented in an enhanced Scheimpflug image (arrow); imbedded image shows axis of the image capture. (C) Typical vesicular lesions with gray halos in the right eye of individual III:3 from family C24 (D) and in the right eye of individual II:1 from family S1 in retroillumination. (E) Specular microscopy imaging of the right eye of individual II:1 from family S1 showing irregular posterior corneal surface and abnormalities of corneal endothelial cell morphology.
Details of systemic features were unavailable as the patient was lost to follow up. Individual II:1 from family B7 (Fig. 1F) reported poor vision since childhood which gradually deteriorated. He was first examined at Moorfields Eye Hospital at the age of 38 years. At the age of 39 years he underwent left PK followed
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by a right PK when he was 41 years old. Except for corneal changes consistent with the diagnosis of PPCD no other abnormalities of the anterior segment were noted. The patient reported poor vision in his father, however, no further details could be obtained. His three siblings had no eyesight problems but were not available for examination. The proband
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I:1/M II:2/M I:1/F II:1/M II:1/M
B5
39 19 32 38 29
44 22 16 10 10 37
Age (yrs)
0.33 0.25 0.1 0.25 0.78
0.66 0.25 0.66 0.5 0.66 CF
RE
0.33 0.33 0.33 0.1 0.6
0.66 0.16 0.66 0.25 0.5 0.66
LE
NA NA NA NA 608
599 601 628 610 666 NA
RE
NA NA NA NA 597
591 609 634 606 657 NA
LE
Thinnest point
Y N N N N Steroid responder N N N N N
Glaucoma
NA −7.00 −1.50 × 10° NA +1.00 NA
NA −8.00 −2.25 × 180° −3.75 −0.25 × 110° −1.75 −2.5 × 180° +0.25 −0.5 × 170° NA
RE
Refraction (DS/DC)
NA −6.00 −0.50 × 90° −1.5 −1.5 × 160° +2.00 NA
NA −3.0 −3.0 × 180° −4,25 −0.5 × 36° −2.75 −2.25 × 180° −0.5 −1.5 × 180° NA
LE
NA 52.7/53.6 PK 46.5/46.9 45.9/48.1
45.2/46.4 50.4/50.8 44.4/44.8 49.5/53.4 44.2/45.7 PK
RE
K1/K2 (D)∗
NA 52.7/52.7 NA 43.5/44.2 47.0/48.4
45.5/46.5 47.7/48.6 45.0/46.9 50.5/54.0 46.0/48.1 PK
LE
BCVA, best corrected visual acuity; CF, counting fingers; D, diopter; DS, diopter sphere; DC, diopter cylinder; F, female; K1, corneal dioptric power in the flattest meridian for the 3 mm central zone; K2, corneal dioptric power in the steepest meridian for the 3 mm central zone; LE, left eye; RE, right eye; M, male; N, no; NA, not available; PK, penetrating keratoplasty; Y, yes;. Yrs, years. Simulated K values and thinnest point were measured in individuals from families C16, C24 and S1 using Pentacam. Topcon OM-4 was used for measurements of K values in subject II:2 from family B4 and probands from families B5 and B6. Abnormal values > 97.5 percentile (>46.1 D for K1, >47.4 D for K2, and >601 μm for thinnest pachymetry [Gilani et al., 2013]) are indicated in bold.
B6 B7 S1
B3
C24
III:1/F IV:1/M II:1/M II:2/M II:3/M II:1/M
Indivi-dual /Gender
C16
Family
BCVA
Table 3 Clinical characteristics of 11 cases with posterior polymorphous corneal dystrophy 3.
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had a history of an enlarged heart since childhood and was indicated for pericardiectomy when he was 37 years old. The proband II:1 from family S1 (Fig. 1F) of Sri Lankan origin had a negative family history for corneal disease although no other family members were available for examination. He was noted to have poor vision at 7 years of age. At the age of 29 years he was suspected to have glaucoma, with IOP measurements of 24 mmHg in the left eye, but with normal optic discs and open anterior chamber angles. Vesicular endothelial lesions (Fig. 2D) and specular microscopy showed enlarged endothelial cells and uneven posterior corneal surface (Fig. 2E). He had a right posterior lamellar keratoplasty with a subsequent BCVA of 0.18 despite a clear graft, suggesting the presence of amblyopia. The patient did not report any general health problems however precise details on extraocular features associated with PPCD3 were not available. The majority (10 out of 16) of patient corneas had steep keratometry values. Pachymetry was increased in 8 out of 12 eyes (Table 3).
Discussion In this study, we report six novel ZEB1 mutations causing PPCD3. We confirm that the disease may present as corneal edema detected at, or soon after, birth (Liskova et al., 2010). In addition, consistent with previous reports, the majority of corneas had an abnormally steep curvature and thickness (Liskova et al., 2010; Raber et al., 2011; Liskova et al., 2013; Aldave et al., 2013). We were also able to compare the phenotype in a pair of male dizygotic twins, who had differences in the degree of corneal steepening and the presence of iris flocculi. To the best of our knowledge this is the first time iris flocculi have been observed in a patient with PPCD3. We suggest that cryptorchidism may also be a part of the PPCD3 phenotype. The prevalence of cryptorchidism in the general population is 1.2% at 1 month of age (Pierik et al., 2005) and 1.1% at 1 year (Berkowitz et al., 1993), while an undescended testicle has to date (including this study) been reported in three of the 50 males with PPCD3 (Krafchak et al., 2005; Aldave et al., 2007; Liskova et al., 2007; Nguyen et al., 2010; Vincent et al., 2009; Lechner et al., 2013; Liskova et al., 2013; Bakhtiari et al., 2013). PPCD has been reported to be caused by mutations in at least three genes (Biswas et al., 2001; Heon et al., 2002; Gwilliam et al., 2005; Krafchak et al., 2005). To date, the majority of Caucasian British PPCD probands (five of seven, 71%) attending Moorfields Eye Hospital have been shown to harbor a causative mutation in ZEB1 coding exons (current study and Liskova et al., 2007). This is in contrast to the reports of US cohorts identifying ZEB1 mutations in approximately 30% of cases (Krafchak et al., 2005; Aldave et al., 2007;
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Bakhtiari et al., 2013), highlighting differences between patient populations. In the Czech Republic, six out of 30 (20%) of probands have a causative mutation in ZEB1 coding exons. This relatively low frequency is likely influenced by the fact that over 50% of Czech PPCD probands are presumed to be descendants of a common founder harboring a mutation within the PPCD1 locus (Liskova et al., 2012). Four of the five novel ZEB1 mutations we identified are predicted to produce a transcript containing a premature termination codon that will be degraded by nonsense mediated decay (NMD) or produce a truncated protein resulting in loss of functional domains, which is consistent with previous studies (Krafchak et al., 2005; Aldave et al., 2007; Liskova et al., 2007; Nguyen et al., 2010; Lechner et al., 2013). It has been estimated that at least 10% of all mutations causing human inherited disease affect pre-mRNA splicing (Krawczak et al., 2007). RNA from the individual with a c.685–2A>G variant was not available for further analysis; therefore, we used in silico splice prediction algorithms to assess its likely effect. Results of the analysis predicted that the mutation would cause aberrant pre-mRNA splicing of the ZEB1 transcript in vivo, and that this would lead to premature termination and either NMD or a truncated protein product. All ZEB1 mutations identified to date are unique to the families, with the exception of the c.1576dup mutation, which has been now observed for the fourth time (Krafchak et al., 2005; Bakhtiari et al., 2013; Lechner et al., 2013), suggesting this may be a mutation hotspot. As a cautionary note, probands from families C16 and B3 were previously reported as ZEB1 negative (Liskova et al., 2007). Reanalysis of the sequence data indicated that their disease-causing changes had been missed in this previous study due to their localization at the end of a PCR fragment, leading to seemingly wild-type sequence assembly. In this study individual I:1 from families C24, B3, B5, B6, and probands from families S1 and B7 did not have a family history of the disease. This may be due to asymptomatic status in one of the parents, disease nonpenetrance or due to the mutation(s) arising de novo. Nonpenetrance and a de novo origin of mutations have been previously documented for PPCD3 (Krafchak et al., 2005; Liskova et al., 2007; Liskova et al., 2010; Bakhtiari et al., 2013). However, because of lack of additional familial samples further molecular genetic investigation could not be performed. Nonocular phenotypes, associated with ZEB1 mutations have been reported previously, most commonly the presence of hernia (Krafchak et al., 2005; Aldave et al., 2007; Lechner et al., 2013; Liskova et al., 2013; Jang et al., 2014). In this study, there was a self-reported history of umbilical hernia in the twins from family C24, and umbilical hernia in the father of the proband from family B3. The possible association of PPCD3 with cryptorchidism warrants further investigation.
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Acknowledgements This work was supported by research grant GACR P301/12/P591 (PL). Institutional support was provided by the PRVOUK-P24/LF1/3 program of the Charles University in Prague. AJH and SJT are supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology. CJE is supported by a Fight for Sight PhD studentship (AJH and SJT). AED is supported by the Lanvern Foundation, Moorfields Eye Charity and Moorfields Special Trustees (AJH and SJT), and the Rosetrees Trust (AED and AJH). LD was supported by the grant SVV-2014–260022.
Conflict of Interest The authors declare no conflict of interest.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Primer sequences used for amplification and sequencing ZEB1. Received: 9 July 2014 Accepted: 30 September 2014
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