Abstract
Purpose:
Familial exudative vitreoretinopathy (FEVR) is a rare hereditary disorder characterized by the failure of peripheral retinal vascularization. The genes FZD4, LRP5, and TSPAN12 are known to be associated with the autosomal inheritance form of FEVR. In this study, we performed mutation screening for FZD4, LRP5, and TSPAN12 in patients with clinical diagnosis of FEVR. In patients with no mutation detected, sequencing analyses for ZNF408, a novel gene potentially related to FEVR, and two other genes related to retinal development, LGR4 and ATOH7, were performed.
Methods:
Mutational studies were done in 51 unrelated patients with diagnosis of FEVR during 2008 to 2012 at the Seoul National University Hospital. These patients were screened previously for NDP gene and confirmed to be negative for mutations. Diagnosis of FEVR was established by ophthalmic examinations. Data collected from medical records included sex, age at diagnosis, clinical presentation, and angiographic findings.
Results:
In this study, we identified 3 known mutations, 10 novel variants with high possibility of pathogenicity, and a whole gene deletion in a total of 18 unrelated patients of 51, resulting in 35.3% of patients being genetically confirmed as having FEVR. Among the patients with pathogenic mutations detected, FZD4 mutations accounted for the largest proportion of autosomal inheritance FEVR cases (13/18 patients, 72.2%), followed by LRP5 (4/18 patients, 22.2%) and TSPAN12 (1/18 patients, 5.6%). No pathogenic mutations were identified in ZNF408, LGR4, and ATOH7. A significant difference in FEVR stage and visual acuity was observed according to the gene involved, showing that patients with FZD4 mutations had milder phenotype.
Conclusions:
Mutations of FZD4 accounted for the largest proportion, which could be directly applied to the testing strategy to start with screening for FZD4 mutations. Panel sequencing consisting of related genes would be an alternative choice for the diagnosis of FEVR. Also, genotype–phenotype correlation suggested in this study could be helpful in genetic counseling of the probands and their family members as well.
Familial exudative vitreoretinopathy (FEVR), first described by Criswick and Schepens in 1969,
1 is a rare hereditary disorder characterized by failure of the peripheral retinal vascularization. Clinical manifestation of the disease can be variable, ranging from nonsymptomatic vascular anomalies in the peripheral retina to bilateral retinal detachments with blindness. The prevalence of this disorder is unknown, but it is likely to be underestimated due to high proportion (79%) of peripheral retinal vascular anomalies detected in asymptomatic family members.
2 Familial exudative vitreoretinopathy does not follow a predictable timeline of progression, occurring throughout childhood and adulthood, and thus, the accurate diagnosis of FEVR is important for long-term monitoring. The condition also is genetically heterogeneous, and found in various modes of inheritance. Autosomal dominant inheritance is the most common form in FEVR, and
FZD4,
LRP5, and
TSPAN12 are the known genes associated with the disease. An additional FEVR-related genetic locus (EVR3) has been mapped to chromosome 11p12–p13, but the gene is not known yet.
3 Genes
LRP5 and
TSPAN12 also are reported to be involved with autosomal recessive inheritance of the disease, usually resulting in more severe phenotype.
4,5 Possibility of autosomal recessive
FZD4 mutations has been mentioned previously,
6,7 but to our knowledge there are no cases with two clear pathogenic mutations reported to date. Additionally, X-linked FEVR is caused by mutations in the
NDP gene, which also is known as a causal gene of Norrie disease. Norrie disease is one of the
NDP-related retinopathies, showing clinically overlapping phenotype with X-linked FEVR.
The four genes known to be associated with FEVR (
NDP,
FZD4,
LRP5, and
TSPAN12) are the essential components of the wingless (Wnt) pathway in the retina. Norrin protein encoded by the
NDP gene, which is not a typical Wnt pathway ligand, binds to the receptor complex of the Wnt pathway composed of the seven-pass transmembrane Frizzled (FZD) receptor along with its coreceptor, low-density lipoprotein receptor-related protein (LRP), and acts as a ligand in the retina.
8 Another causative gene,
TSPAN12, also is known to form a receptor complex with
FZD4 and
LRP5. Pathogenic mutations affecting the function of these genes results in abnormal retinal vascular formation.
Considering that the existing proportion of mutation detection does not generally exceed 50%,
2 searching for novel causative genes of FEVR should be continued to increase the proportion of molecularly confirmed patients. In this study, we chose several other genes as FEVR-related candidates. One of them was
ZNF408, which was suggested recently to be associated with autosomal dominant FEVR.
9 Though it has not been explained how this gene acts along with other components of the Norrin pathway, mutants of this gene resulted in abnormal retinal vasculogenesis, suggesting that
ZNF408 might account for a considerable proportion of FEVR. Another gene,
LGR4, has been reported previously to act as a receptor for Norrin, with a high possibility of having a role in the Wnt signaling pathway related to retinal vascularization.
10,11 Additionally,
ATOH7, mutations of which originally were reported in patients with persistent hyperplastic primary vitreous (PHPV), also was included due to the phenotype of PHPV resembling FEVR and also the role of
ATOH7 in retinovascular disease.
12
Here, we show mutation spectrum of FZD4, LRP5, and TSPAN12 in patients with clinical diagnosis of FEVR, who had been confirmed previously as negative for NDP mutations. Mutation screening for ZNF408, LGR4, and ATOH7 genes also was performed in patients with no detected mutation in three autosomal inheritance FEVR-related genes. Then, we reviewed genotype–phenotype correlation of each gene in molecularly confirmed patients.
A retrospective chart review was performed in patients with a diagnosis for FEVR between January 2008 and December 2012 at the Seoul National University Children's Hospital. The diagnostic criteria for FEVR composed of three of the following: birth at full term or premature birth with no evidence of retinopathy of prematurity, a presence of peripheral retinal avascular area, and variable degree of nonperfusion, vascular leakage, or retinal neovascularization in fluorescein angiography. Patients were included if a final diagnosis of FEVR was given after the complete ophthalmic examination, including fundus examination and fluorescein angiography using Retcam (Clarity Medical Systems, Inc., Pleasanton, CA, USA).
Among the patients diagnosed with FEVR by clinical diagnostic criteria, patients with NDP mutations were excluded and only those confirmed as negative for NDP mutation were included. Mutational studies were done in 51 unrelated patients who agreed on the genetic testing and, among them, three had another family member with the diagnosis of FEVR. When these patients were confirmed with a mutation, family members also were tested. Informed consent was obtained from all individuals after explaining the nature and possible consequences of the study. Experiments were performed according to the Declaration of Helsinki and were approved by the hospital's ethics committee (IRB No. 1503-036-654).
Clinical characteristics, including mainly angiographic findings, the presence of macular ectopia, macular dragging, retinal folds, and patient demographics, including sex, age at presentation, and cycloplegic refractive errors (spherical equivalent) at last follow up visit were analyzed in patients with FEVR.
The existing proportion of FEVR patients who are genetically diagnosed by the detection of pathogenic mutations is approximately 40% to 50%.
2 Although the contribution of each gene to this disease differs between study populations, it is known that 4% to 40% is attributed to
FZD4, 12% to 25% to
LRP5, and 3% to 10% to
TSPAN12.
22–28 In this study, pathogenic mutations were detected in 18 patients, resulting in 35.3% of patients being genetically confirmed as having FEVR. Two recurrent mutations, c.313A>G and c.1282_1285delGACA in
FZD4 were detected in our patient group. Allele c.313A>G in
FZD4, which was detected in four unrelated patients in this study, has been reported previously in Japanese and Chinese FEVR patients, suggesting that this mutation occurs frequently in the Asian population. Haplotype analysis indicated that this mutation was less likely to be derived from a common founder. Among 18 patients with pathogenic mutation, 13 had
FZD4 mutations, showing that mutations in this gene comprise the highest proportion of autosomal inheritance FEVR in Korean population (13/51 patients, 25.5%). This proportion was similar to the previously reported 20%.
6,19 The proportion of
LRP5 mutations contributing to FEVR in our study group seems lower than reported proportion of other studies.
22,26,29
Interestingly, our study showed that carrying a mutation in FZD4 seemed to result in milder phenotype than LRP5. Despite the limitation of small number of molecularly confirmed patients, those with FZD4 mutations showed lower FEVR stages and better visual acuity compared to those with LRP5 mutations. Though the phenotype of FEVR has long been considered to be indistinguishable by gene involved, analyzing a larger population of molecularly confirmed patients actually might be able to suggest the genotype–phenotype correlation, to the level further than currently known phenotypes of osteopenia or osteoporosis in the patients with LRP5 mutations.
Additionally, there was a suspected case of a gross gene deletion in TSPAN12, which was not confirmed in the present study. A symptomatic subject with a homozygous variant of c.484G>A in TSPAN12 did not segregate with the proband's symptomatic brother who had homozygous G. The homozygous state of this rare variant may imply gross deletion of the gene, but a clearer explanation will be provided when gene dosage analysis for TSPAN12 is performed.
Overall, pathogenic mutations were not detected in more than half of the patients in this study. No significant mutations were detected in the novel causative gene ZNF408, or in the possibly related LGR4 and ATOH7 genes. In cases where no mutation was found, further analysis to determine the novel gene contributing to FEVR still is needed. Targeting Wnt pathway-related genes would be an effective strategy. Additionally, mutations in the yet to be defined gene EVR3 also may contribute to the disease.
In conclusion, we showed the mutation spectrum of three genes: FZD4, LRP5, and TSPAN12 in Korean FEVR patients. Mutations of FZD4 were the most common in cases of autosomal inheritance FEVR. Thus, a testing strategy for FEVR starting with screening for FZD4 mutations should be applied clinically. Panel sequencing consisting of the genes mentioned above is an alternative choice. Genetic counseling of a proband and asymptomatic family members also would be helpful in further management and prevention of the disease. Further analyses of novel causative genes and genotype–phenotype correlations may contribute to a better understanding of the pathophysiological consequences of FEVR.
The authors alone are responsible for the content and writing of the paper.
Disclosure: S.H. Seo, None; Y.S. Yu, None; S.W. Park, None; J.H. Kim, None; H.K. Kim, None; S.I. Cho, None; H. Park, None; S.J. Lee, None; M.-W. Seong, None; S.S. Park, None; J.Y. Kim, None