Prevalence and Implications of TERT Promoter Mutation in Uveal and Conjunctival Melanoma and in Benign and Premalignant Conjunctival Melanocytic Lesions

Abstract

Purpose.: Hot-spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in cutaneous and conjunctival melanoma and are exceedingly rare in uveal melanoma. No information is available on the presence of these mutations in the conjunctival melanocytic precursor lesion primary acquired melanosis (PAM). We tested a cohort of uveal and conjunctival melanomas as well as conjunctival benign and premalignant melanocytic lesions for TERT promoter mutations in order to elucidate the role of these mutations in tumor progression.

Methods.: TERT promoter mutation analysis on fresh tumor DNA and DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis in 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi.

Results.: Mutations of the TERT promoter were not identified in conjunctival nevi or PAM without atypia, but were detected in 2/25 (8%) of PAM with atypia and 16/39 (41%) of conjunctival melanomas. A single TERT promoter mutation was detected in 102 uveal melanomas (1%).

Conclusions.: We present the second documented case of TERT promoter mutation in uveal melanoma. In comparison with other types of melanoma, TERT promoter mutations occur at extremely low frequency in uveal melanoma. TERT promoter mutations are frequent in conjunctival melanoma and occur at lower frequency in PAM with atypia but were not detected in benign conjunctival melanocytic lesions. These findings favor a pathogenetic tumor progression role for TERT promoter mutations in conjunctival melanocytic lesions.

Introduction

Melanoma is one of the most deadly malignancies of the eye and ocular surface. The incidence of uveal melanoma (5.6 per million) has remained unchanged for decades, ¹ but the incidence of conjunctival melanoma, although rare (0.45–0.8 per million), ² shows a trend of increasing frequency of diagnosis in Europe and the United States. ^3,4 Telomerase reverse transcriptase (TERT) promoter mutations occur frequently in cutaneous melanoma ^5,6 and in conjunctival melanoma ⁷ but rarely in cutaneous acral melanoma ⁸ and uveal melanoma. ^7,9 The frequency of TERT promoter mutations in primary acquired melanosis (PAM) and conjunctival nevi has not yet been described. Here, we investigated the presence of TERT promoter mutations in uveal melanoma, conjunctival melanoma, PAM with atypia, PAM without atypia, and conjunctival nevi in order to elucidate the role of these mutations in tumor progression. 

Materials and Methods

Patients and Samples

Tissue specimens were obtained from patients with a conjunctival melanocytic lesion or uveal melanoma who had undergone biopsy, excision, or enucleation between 1972 and 2013 at the Erasmus University Medical Center or The Rotterdam Eye Hospital, The Netherlands. Fresh tissue samples were obtained from enucleation specimens of 102 primary uveal melanomas. Conjunctival tissue of all patients diagnosed with conjunctival melanoma or PAM (n = 205), as well as 56 patients with conjunctival nevi, was selected from the electronic archives of the Department of Pathology. Paraffin-embedded tissues were retrieved from the archive, and the amount of tissue present in the blocks was assessed. Samples of 125 patients contained enough material for further study. Next the original glass slides were retrieved from the archive, and the initial diagnoses were reassessed by a pathologist specialized in ophthalmic pathology (RMV), as well as mitosis count, Breslow's thickness, presence of leukocyte infiltration, and necrosis (although rare in melanoma). After confirming diagnosis and assessment, 56 conjunctival nevi samples, 14 PAM without atypia samples, 26 PAM with atypia samples, and 39 conjunctival melanoma samples were used for DNA isolation. Clinical details were obtained from patient records. The study was performed in accordance with the tenets of the Declaration of Helsinki. 

DNA Isolation

DNA from uveal melanomas was extracted directly from fresh tumor tissue or frozen tumor using the QIAmp DNA-mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA isolation procedure for the conjunctival nevi, PAM, and conjunctival melanoma samples was performed on formalin-fixed, paraffin-embedded (FFPE) specimens. Criterion for DNA collection was the presence of more than 70% tumor cells. For PAM samples this percentage was not feasible, and a minimum of 20% tumor cells was used for these cases. Microdissection was performed by scraping the tumor cells manually from hematoxylin-stained sections on a glass slide. DNA was extracted from the tissue fragments by incubation overnight at 56°C in lysis buffer (A7933; Promega, Leiden, The Netherlands) containing 2 mg/mL proteinase K and 5% Chelex 100 resin. Proteinase K was inactivated at 100°C for 10 minutes, and the DNA was separated from cell debris by centrifugation at maximum speed in a microcentrifuge for 5 minutes. 

Chromosomal and Gene Analyses in Uveal Melanoma

Copy number variations in uveal melanoma on chromosomes 1, 3, 6, and 8 were investigated by fluorescence in situ hybridization (FISH) and single nucleotide polymorphism analysis, as described previously. ¹⁰ The GNAQ and GNA11 mutation status was determined with Sanger sequencing, ¹¹ and BAP1 expression was examined as reported previously. ¹²  

Mutation Analysis

SNaPshot analysis to determine the presence of mutations in three mutational hot spots, C228, C242, and C250 (chr5:1,295,228C>T; chr5:1,295,242-243CC>TT; chr5:1,295,250 C>T, respectively; hg19), in the promoter region of TERT was performed essentially as described previously. ¹³ A 155-bp DNA segment was amplified with upstream primer 5′-AGCGCTGCCTGAAACTCG-3′ and downstream primer 5′-CCCTTCACCTTCCAGCTC-3′ as described above. Subsequently, a single nucleotide probe extension assay was performed with probes designed to anneal to the sites of interest (228 probe: 5′-T23 GGCTGGGAGGGCCCGGA-3′; 242-243 probe: 5′-T27GGAGGGGGCTGGGCGG-3′; 250 probe: 5′-T39 CTGGGCCGGGGACCCGG-3′). These probes were adapted with poly T tails of different lengths to allow separation of the extended products by size. SNaPshot analysis was performed with the SNaPshot multiplex kit (Life Technologies, City, State, Country) according to manufacturer's instructions. Thermal cycler conditions were as follows: 35 cycles of 10 s at 96°C and 40 s at 58.5°C. SNaPshot products were analyzed on an ABI Prism 3730xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). 

Statistical Analyses

All statistical analyses were performed with the Statistical Package for Social Science (IBM SPSS Statistics 20, Chicago, IL, USA). P values equal to or below 0.05 were considered to be statistically significant. Correlations were calculated with either the χ² test or Fisher's exact test (categorical variables) or the Mann-Whitney U test (continuous variables). Kaplan-Meier survival curves were made for survival analysis by using the log rank statistic. 

Results

Study Cohort

SNaPshot analyses were obtained from the tumor samples of 102 uveal melanomas, 39 conjunctival melanomas, 26 PAM with atypia, 14 PAM without atypia, and 56 conjunctival nevi patients. The samples were primary or recurrent tumors, and seven cases were tested from both primary tumor and recurrences, with identical results. In two PAM cases no reliable results could be obtained because of insufficient quality of the DNA isolated from FFPE tissue. The uveal melanoma group consisted of 55 men and 47 women with a mean age at diagnosis of 61.5 years (range, 21–86 years). Ninety-one tumors were localized in the choroid and 11 in the ciliary body. The mean largest tumor diameter and prominence was 13.4 and 8.2 mm, respectively. Thirty-two of 76 (32.4%) investigated uveal melanomas showed an absent BAP1 expression, and 93 of 99 (91.2%) tumors harbored a mutation in either GNAQ or GNA11. The clinical, histopathologic, and molecular characteristics of the uveal melanomas are shown in Table 1. The clinical and histopathologic characteristics of the conjunctival melanocytic lesions are listed in Table 2. The mean age at diagnosis was 41.3, 48.0, 64.0, and 62.5 years for conjunctival nevi, PAM without atypia, PAM with atypia, and conjunctival melanoma, respectively. Recurrences of the melanocytic lesions occurred predominantly in the PAM with atypia and conjunctival melanoma group (57.7% and 48.7%, respectively). No metastases were observed in patients with conjunctival nevi or PAM without atypia. Conjunctival nevi were located at different sites on the conjunctiva, while PAM without atypia was located in the bulbar conjunctiva in 71% of cases and more specifically at the limbus in another 21%. Of the PAM lesions with atypia, 17 were located at the limbus (65%), 3 on the palpebral conjunctiva, 1 on the fornix, 1 on the bulbar conjunctiva, and 1 on the caruncula lacrimalis, and 3 were multifocal. Eighteen (46%) conjunctival melanomas were located at the limbus, 7 on the bulbar conjunctiva, 9 on the palpebral conjunctiva, and 2 in the fornix. The mean Breslow thickness of conjunctival melanomas was 2.3 mm (range, 0.3–17.0 mm). 

Table 1

 

Clinical, Pathologic, and Genetic Characteristics of the Uveal Melanoma Cohort, n = 102

Uveal Melanoma	n = 102
Clinical features
Sex
Male, n (%)	55 (53.9)
Female, n (%)	47 (46.1)
Mean age at diagnosis, y (range)	61.5 (21–86)
Metastasis
Present, n (%)	46 (45.1)
Not present, n (%)	56 (54.9)
Survival
Alive, n (%)	35 (34.3)
Death due to metastasis, n (%)	44 (43.1)
Death due to other cause, n (%)	23 (22.5)
Mean metastasis-free survival, mo (range)	78.3 (0.8–209.1)
Histopathologic features
Location
Choroid, n (%)	91 (89.2)
Ciliary body, n (%)	11 (10.8)
Mean largest tumor diameter, mm (range)	13.4 (6.0–22.0)
Mean prominence, mm (range)	8.2 (1.5–22.0)
Cell type
Spindle, n (%)	45 (44.1)
Mixed/epithelioid, n (%)	57 (55.9)
Extracellular matrix patterns
Present, n (%)	49 (48.0)
Not present, n (%)	52 (51.0)
Lymphocytic infiltration
Present, n (%)	6 (5.9)
Not present, n (%)	31 (30.4)
Not examined, n (%)	65 (63.7)
Necrosis
Present, n (%)	22 (21.6)
Not present, n (%)	16 (15.7)
Not examined, n (%)	64 (62.7)
Extraocular extension
Present, n (%)	12 (11.8)
Not present, n (%)	90 (88.3)
BAP1 expression
Negative, n (%)	32 (31.4)
Positive, n (%)	44 (43.1)
Genetic features
Chromosome 3
Loss, n (%)	52 (51.0)
Normal, n (%)	49 (48.0)
Chromosome 8p
Loss, n (%)	21 (20.6)
Normal, n (%)	61 (59.8)
Gain, n (%)	19 (18.6)
Chromosome 8q
Loss, n (%)	1 (1.0)
Normal, n (%)	37 (36.3)
Gain, n (%)	63 (61.8)
GNAQ or GNA11 mutation
Mutated, n (%)	93 (91.2)
Wild type, n (%)	6 (5.9)

Table 2

 

Clinical, Pathologic, and Molecular Characteristics of the Conjunctival Lesion Cohort, n = 135

	Conjunctival Nevi, n = 56	PAM Without Atypia, n = 14	PAM With Atypia, n = 26	Conjunctival Melanoma, n = 39
Clinical features
Sex
Male, n (%)	27 (48.2)	3 (21.4)	13 (50.0)	26 (66.7)
Female, n (%)	29 (51.8)	11 (78.6)	13 (50.0)	13 (33.3)
Mean age at diagnosis, y (range)	41.3 (5–87)	48.0 (17–85)	64.0 (34–87)	62.5 (16–85)
Recurrence
Yes, n (%)	0	1 (7.1)	15 (57.7)	19 (48.7)
No, n (%)	56 (100.0)	12 (85.7)	10 (38.5)	20 (51.3)
Metastasis
Present, n (%)	0	0	2 (7.7)	8 (20.5)
Not present, n (%)	52 (92.9)	13 (92.9)	22 (84.6)	25 (64.1)
Survival
Alive, n (%)	49 (87.5)	11 (78.6)	17 (65.4)	24 (61.5)
Death due to metastasis, n (%)	0	0	1 (3.8)	3 (7.7)
Death due to other cause, n (%)	2 (3.6)	0	6 (23.1)	4 (10.3)
Lost to follow-up, n (%)	5 (8.9)	3 (21.4)	2 (7.7)	8 (20.5)
Mean follow-up, mo (range)	60.6 (6.9–249.5)	142.0 (2.6–219.5)	83.0 (1.2–225.5)	66.6 (1.0–322.8)
Histopathologic features
Location
Palpebral conjunctiva, n (%)	7 (12.5)	1 (7.1)	3 (11.5)	9 (23.1)
Fornix conjunctiva, n (%)	1 (1.8)	0	1 (3.8)	2 (5.1)
Bulbar conjunctiva, n (%)	23 (41.1)	10 (71.4)	1 (3.8)	7 (17.9)
Limbus, n (%)	7 (12.5)	3 (21.4)	17 (65.4)	18 (46.2)
Caruncula lacrimalis, n (%)	14 (25.0)	0	1 (3.8)	0
Tumor size
Mean Breslow thickness, mm (range)	-	-	-	2.3 (0.3–17.0)
Cell type
Stromal, n (%)	16 (28.6)	-	-	-
Compound, n (%)	40 (71.4)	-	-	-
Spindle, n (%)	-	-	-	9 (23.1)
Mixed spindle/epithelioid, n (%)	-	-	-	8 (20.5)
Epithelioid, n (%)	-	-	-	17 (43.6)
Ulceration
Present, n (%)	-	-	-	12 (30.8)
Not present, n (%)	-	-	-	20 (51.3)
Erosion
Present, n (%)	-	-	-	3 (7.7)
Not present, n (%)	-	-	-	29 (74.4)
Necrosis
Present, n (%)	-	-	-	2 (5.1)
Not present, n (%)	-	-	-	30 (76.9)
Vasoinvasive
Yes, n (%)	-	-	-	1 (2.6)
No, n (%)	-	-	-	30 (76.9)
Inflammation
Present, n (%)	-	-	-	22 (56.4)
Not present, n (%)	-	-	-	10 (25.6)
Focality
Unifocal lesion, n (%)	11 (19.6)	8 (57.1)	6 (23.1)	14 (35.9)
Multifocal lesion, n (%)	0	2 (14.3)	15 (57.7)	17 (43.6)

SNaPshot TERT Promoter Mutation Analysis

One of 102 uveal melanomas (1.0%) showed a 250C>T TERT promoter mutation. This tumor had a disomy of chromosomes 1p and 3, and a gain of chromosomes 6 and 8. In addition, this sample harbored a heterozygous GNA11 mutation in exon 5 (p.Q209L) and had normal BAP1 staining, consistent with a primary low-risk uveal melanoma signature. Blood of this patient was available for TERT analyses revealing a wild-type status. A total of 16 TERT promoter mutations, three 228C>T, two 242CC>TT, and eleven 250C>T, were found in the 39 conjunctival melanomas (41.0%); and 2 of 25 (8.0%) PAM with atypia showed a mutation, one 228C>T (Fig. 1) and one 250C>T (Fig. 2, Table 3). In one case a conjunctival nevus and a conjunctival melanoma were analyzed from the same patient as unrelated lesions. In seven cases, concurrent PAM and conjunctival melanoma were analyzed from the same patient. In none of these cases did the lesion contain a TERT promoter mutation. In the two cases of PAM with atypia that carried TERT promoter mutations, one recurred once as a PAM with atypia and was then radically excised; the other case also had one recurrence as PAM with atypia, which was irradically excised. Subsequently, both these patients received successful additional treatment with cryotherapy and topical mitomycin C with no recurrences. No TERT promoter mutations were identified in 13 PAM without atypia and 56 conjunctival nevi patients (16 stromal and 40 compound nevi). 

Figure 1

 

Histology and SNaPshot analysis results of case 15, PAM with severe atypia (A), 228C>T mutation (B) indicated by arrow.

Figure 2

 

Histology and SNaPshot analysis results of case 72, PAM with moderate atypia (A), 250C>T mutation (B) indicated by arrow.

Table 3

 

Overview of the TERT Promoter Mutations Found in Different Types of Melanocytic Conjunctival and Uveal Lesions

	Conjunctival Nevi, n (%)	PAM Without Atypia, n (%)	PAM With Atypia, n (%)	Conjunctival Melanoma, n (%)	Uveal Melanoma, n (%)
TERT mutation	0/56 (0)	0/13 (0)	2/25 (8.0)	16/39 (41.0)	1/102 (1.0)
g.1295228C>T	-	-	1	3	0
g.1295242CC>TT	-	-	0	2	0
g.1295250C>T	-	-	1	11	1

Correlations of TERT Promoter Mutation Status With Clinicopathologic Patient Characteristics

The mean age of patients with conjunctival nevi did not differ significantly from that of patients with PAM without atypia (41.3 vs. 48.0 years, P = 0.284). Patients with PAM with atypia and conjunctival melanoma were significantly older than patients with PAM without atypia (64.0 vs. 48.0, P = 0.025; 62.5 vs. 48.0 years, P = 0.017). No significant difference in mean age was observed between PAM with atypia and conjunctival melanoma (64.0 vs. 62.5 years, P = 0.708). Overall, TERT mutations did not significantly correlate with the development of metastatic disease (P = 1.000). For subgroup analysis, no correlations could be calculated for uveal melanoma. The conjunctival melanoma cases with TERT promoter mutation did not show a statistically significant correlation with adverse histologic prognostic factors when compared to nonmutated cases as to mean Breslow thickness (2.7 vs. 2.1 mm, respectively, P = 0.824), mitotic count (14.6 vs. 7.3, respectively, P = 0.138), or necrosis (P = 0.502). There was also no significant association between conjunctival location and the presence of a TERT promoter mutation in PAM with atypia and conjunctival melanomas (P = 0.153). No significant differences in clinical outcome were observed. The mean survival of TERT promoter mutated cases when compared to nonmutated cases did not differ significantly (75.3 vs. 60.4 months, respectively, P = 0.490). Six of 16 patients with a TERT mutated tumor developed a recurrence of the melanocytic lesion after excision compared to 13 of 23 patients with a wild-type tumor (P = 0.272, Fig. 3A). In total, 8 of 39 patients with conjunctival melanoma developed metastatic disease; four of these tumors harbored a TERT mutation (P = 0.527, Fig. 3B). Three out of 16 mutated conjunctival melanoma cases died from metastatic disease compared to none of the wild-type cases. 

Figure 3

 

Kaplan-Meier survival estimate for the time to recurrence of conjunctival melanoma (A) and the time to metastasis of conjunctival melanoma (B), showing no significant differences in survival between the TERT mutated and wild-type cases.

Discussion

Melanomas are the most common lethal primary ocular cancers. In the ophthalmic practice, melanomas mainly occur in two types, uveal melanoma and conjunctival melanoma, which differ in both genetic and molecular background. We detected TERT promoter mutations in a high percentage (41%) of conjunctival melanomas as compared to only 1% of uveal melanomas. These results are in accordance with one other cohort in which TERT promoter mutations were detected in approximately one-third of conjunctival melanomas. ⁷ We present the second case of a TERT promoter mutation in an otherwise cytogenetically and molecularly unremarkable case of uveal melanoma. Both the TERT promoter mutated uveal melanoma described here and the TERT promoter mutation-positive uveal melanoma described by Dono et al. ⁹ have a cytogenetic low risk signature. Until now, TERT promoter mutations have been detected in only 1 of a total of 97 uveal melanoma-derived samples from two different cohorts. ^7,9 Uveal melanomas, in contrast to conjunctival melanomas, lack BRAF or NRAS mutations but frequently have GNAQ, GNA11, BAP1, or SF3B1 mutations. ^9,11 These findings confirm that since genetic alterations are different, ¹⁴ the molecular pathogenesis of uveal melanoma is distinct from that of conjunctival or cutaneous melanoma. In contrast to earlier reports, the mere presence of TERT promoter mutations cannot be used to distinguish primary uveal melanoma from (metastatic) cutaneous or conjunctival lesions. ⁷  

The core promoter region of the TERT gene encodes the rate-limiting catalytic reverse transcriptase subunit of the telomerase ribonucleoprotein complex. The g.1295228C>T, g.1295250C>T, or less common g.1295242CC>TT mutations upstream of the transcription start site create de novo CCGGAA/T general binding motifs for E-twenty six/ternary complex factor (Ets/TCF) transcription factors, which differs from preexisting GGAA/T Ets binding sites within the TERT promoter. The Ets transcription factors are downstream targets of RAS-RAF-MAPK pathways, and TERT promoter mutations are suggested to have synergistic effects with activating BRAF or NRAS mutations to promoter tumor cell proliferation. BRAF and NRAS mutations have been proposed to be driver mutations in the development of cutaneous melanocytic neoplasms since they are present in both benign and malignant neoplastic lesions. ^15,16  

We are the first to show the presence of TERT promoter mutations in a premalignant melanocytic lesion, PAM with atypia, and its absence in PAM without atypia and conjunctival nevi. The mutation frequency of 33% reported in primary cutaneous melanomas ^6,17 is similar to the mutation rate in primary conjunctival melanomas. Conjunctival melanomas, like cutaneous melanomas, frequently harbor BRAF or NRAS mutations. ⁷ The high frequency of TERT promoter mutations and the higher than expected rate of concomitant mutations in the TERT promoter and BRAF in cutaneous melanoma ⁶ might suggest that TERT promoter mutations are driver mutations in the pathogenesis of melanoma. However, in contrast to BRAF mutations, TERT promoter mutations were not identified in benign cutaneous nevi (n = 34), ^6,17 nor in conjunctival nevi ⁷ or PAM without atypia. The possible role of TERT promoter mutations in molecular progression is demonstrated by the identification of these mutations in a low percentage of premalignant melanocytic lesions, PAM with atypia, and in a higher percentage in conjunctival melanoma. In the past, it has been observed that atypia does lead to a highly increased chance for malignant progression. Although our cohort contained only 14 cases of PAM without atypia, this would be in agreement with that observation. The acquisition of TERT promoter mutations can be hypothesized to facilitate stabilization of the transformed genome through reversal of telomeric loss. Many conjunctival melanocytic nevi carry BRAF mutations, ¹⁸ whereas TERT promoter mutations are detected only in primary melanoma and, as we have shown, in PAM with atypia. In cutaneous squamous cell carcinoma lesions a similar role for TERT promoter mutations can be observed, with mutations in 50% of squamous cell carcinomas and 9% of Bowen's disease (in situ lesions). In contrast, the role for TERT promoter mutations as a driver mutation is more convincing in urothelial cell neoplasms, where TERT promoter mutations are present in a high percentage (74%) in both low-grade and high-grade in situ urothelial cell carcinoma ^13,19 as well as in invasive urothelial cell carcinoma (53%). ^13,20  

The increased telomerase expression associated with TERT promoter mutations ⁵ offers new therapeutic possibilities in TERT mutated conjunctival melanoma. General reverse transcriptase inhibitors such as AZT (azidothymidine), which acts on the reverse transcriptase activity of TERT, may warrant investigation for potential therapeutic potential in conjunctival melanoma with TERT promoter mutations. ²¹ Another way to inhibit telomerase function is to reduce levels of TERT transcripts. The telomerase nucleotide bases inhibitor, imetelstat (GRN163L), has shown effectiveness in melanoma cell lines ²² and entered phase I ²³ and II clinical trials for other tumor types. More specific chemical inhibitors of telomere extension, MST-312, BIBR1532, b-rubromycin, PIPER {N,N0-bis [2-(1-piperidino) ethyl]-3,4,9,10-tetracarboxylic diimide}, and TmPyP4 are in an experimental phase at this moment. ²⁴  

In conclusion, we present the first data on TERT promoter mutations in PAM with atypia and propose its possible role as a molecular tumor progression marker for conjunctival melanocytic lesions. 

Acknowledgments

Supported by Grants SNOO2012-21 and SNOO2012-13 from the Stichting Nederlands Oogheelkundig Onderzoek. 

Disclosure: A.E. Koopmans, None; K. Ober, None; H.J. Dubbink, None; D. Paridaens, None; N.C. Naus, None; S. Belunek, None; B. Krist, None; E. Post, None; E.C. Zwarthoff, None; A. de Klein, None; R.M. Verdijk, None 

Footnotes

 See the appendix for the members of the Rotterdam Ocular Melanoma Study Group.

Appendix

ROMS: Rotterdam Ocular Melanoma Study Group

Rotterdam Eye Hospital: A. Dion A. Paridaens, Ophthalmologist, MD, PhD; Hanneke W. Mensink, Ophthalmologist, MD, PhD; Thomas van den Bosch, Resident Ophthalmology, MD, PhD 

Department of Ophthalmology, Erasmus MC: Nicole C. Naus, Ophthalmologist, MD, PhD; Emine Kilic, Ophthalmologist, MD, PhD; Anna E. Koopmans, Resident Ophthalmology, MD, PhD; Jackelien G. van Beek, Ophthalmologist, MD 

Department of Pathology, Erasmus MC: Robert M. Verdijk, Ophthalmic Pathologist, MD, PhD 

Department of Clinical Genetics, Erasmus MC: Annelies de Klein, Clinical Cytogeneticist, PhD; Serdar Yavuzyigitoglu, PhD Student; Jolanda Vaarwater, Laboratory Technician