Introduction

Familial exudative vitreoretinopathy (FEVR) is a congenital retinal vascular disease characterized by retinal nonperfusion, proliferative retinopathy, and retinal detachment.¹ Clinically, FEVR displays remarkably varying phenotypes, from barely detectable retinal vascular anomaly to retinal detachment.² Classical clinical findings consist of peripheral avascular retina, neovascularization, exudation, dragging of vascular elements, and retinal detachment.¹ Mutations of the Wnt/Norrin signaling pathway underlie the molecular mechanisms leading to FEVR.³ Particularly, four genes are well documented to be related to FEVR: FZD4, LRP5, TSPAN12, and NDP.^3,4,5,6

Hedgehog (Hh) signals are considered to play an important role in embryonic gut development. Abnormal Hh signaling had been identified in the molecular etiology of the common gastrointestinal tract malformations (GTMs).⁷ The family of Hh signaling proteins includes three members: sonic (Shh), Indian (Ihh), and desert (Dhh) hedgehogs. Shh signaling forms the key molecules in gut morphogenesis. These molecules are expressed in the endoderm throughout early embryogenesis and have an important role in gut formation.⁸

In addition to gut development, the Shh signaling pathway is important in neuronal differentiation, axon guidance, and angiogenesis.⁹ Astrocytes secrete Shh to promote formation of the blood-brain barrier.¹⁰ A recent study with an in vitro model of retinal vascular endothelial cells demonstrated that the Shh pathway regulates endothelial permeability.¹¹ A mutation in the Shh pathway may cause a disorder consisting of GTMs and retinal vascular disease.

The aim of this study was to report three unrelated sporadic patients with GTMs exhibiting the FEVR phenotype. Genetic analyses revealed defects in the Shh pathway. This association may necessitate the referral of patients with GTMs to retina specialists.

Materials and Methods

This was a case series including six eyes of three neonates who underwent routine ophthalmological examination during their hospitalization in the neonatal surgical intensive care unit for GTMs between September 2016 and September 2019. All three of the neonates were male, having gestational ages of 39, 38, and 39 weeks and birth weight of 3500, 3600, and 3300 grams, respectively (Table 1). All were examined by the same ophthalmologist (N.S.K.) using a binocular indirect ophthalmoscope and diagnosed as having FEVR. FEVR was classified based on ophthalmoscopic findings: stage 1, avascular periphery; stage 2, avascular periphery with neovascularization; stage 3, macula-sparing retinal detachment; stage 4, macula-involving retinal detachment; and stage 5, complete retinal detachment.¹²

Diode laser photocoagulation was performed to 360° presumed avascular areas of retina (0.15 seconds and 160 mW power) in a near confluent pattern, under general anesthesia, in the operating room.

The research was conducted at our tertiary care facility after receiving approval from the local ethics committee (KA19/306). The study complied with the principles of the Declaration of Helsinki. Informed consent form was obtained from the parents of the patients.

Statistical Analysis

Patient DNA was isolated from whole blood (3 mL) using the QIA amp DNA blood kit (QIAGEN, Hilden, Germany). Three micrograms of high-molecular-weight DNA was sheared using a Covarias ultrasonicator and the genomic DNA library was prepared with an Agilent SureSelect Target Enrichment System. Following enrichment with Agilent SureSelect Human All Exon V7, sequencing was performed on an IlluminaHiSeq 2500 System (Illumina, San Diego, USA). Data interpretation and variant calling were performed on SOPHiA DDM® version 5.4.2 (Sophia Genetics, Switzerland).

Results

Fundus examination revealed flat neovascularizations and brush-paint-like retinal hemorrhages in all 6 eyes. Figure 1a shows a macular image from patient 1 and Figure 1b shows a presumed avascular area and hemorrhage in the same patient. Fundus images of patient 2 before and after treatment are shown in Figure 1c and 1d. Patient 3 also had hemorrhages as a result of neovascularizations on the border of the vascular zone and presumed avascular retinal regions (Figure 1e,f). All eyes were diagnosed as having stage 2 FEVR without exudation (stage 2A). Presumed avascular areas were treated successfully with laser photocoagulation and no additional treatment was needed. Recurrence was not seen in any case.

Two of the neonates had anal atresia and one had Hirschsprung disease (HSCR). Next, we explored the putative causative mutations associated with the patients’ phenotypes. We identified a heterozygous LRP5 (low-density lipoprotein receptor-related protein 5) mutation (rs80358313, NP_002326.2:p.Gly610Arg, NM_002335.4:c.1828G>A) in patient 1 and a heterozygous KIF7 (kinesin-like protein KIF7) mutation (rs143877028, NP_940927.2:p.Ser1281Ile, NM_198525.3:c.3842G>T) in patient 2, who both presented with anal atresia. In patient 3, who had HSCR, we detected a heterozygous GLI2 (zincfinger protein GLI2) mutation (rs751513015, NP_005261.2:p.Asp705Asn, NM_005270.4:c.2113G>A). Altogether, our findings demonstrated mutations of different genes in the Shh pathway.

Discussion

Familial exudative vitreoretinopathy was first defined by Criswick and Schepens¹ in 1969. FEVR presents with varying clinical manifestations but is limited to abnormalities in ocular development.¹³ Four genes were found to be associated with FEVR. Autosomal dominant FEVR appears from mutations in FZD4, LRP5, and TSPAN12; the autosomal recessive form occurs in homozygous LRP5 mutations, and X-linked FEVR arises from mutations in NDP.⁶ Norrie disease is another hereditary disorder occurring mainly in full-term infants, defined by abnormal retinal vascularization causing retinal detachment. Norrie disease causes blindness soon after birth and most patients have systemic disorders such as mental retardation and deafness.¹³ Mutations in the Norrie disease protein (NDP) gene leads to Norrie disease.¹⁴ At birth, NDP is markedly increased in the retina in order to enhance levels of norrin, which is involved in progenitor proliferation in the postnatal period.¹⁵ McNeill et al.¹⁶ showed that Hh signaling regulates NDP expression and concluded that NDP is essential downstream of Hh activation to trigger retinal progenitor proliferation in the retina. In addition, Wang et al.¹⁷ showed that Hh signaling is mandatory to support retinal precursor cell proliferation.

Surace et al.¹⁸ investigated the association between the Hh pathway and retinopathy of prematurity (ROP) in murine models and found upregulation of Hh expression similar to vascular endothelial growth factor (VEGF). To support the results, the investigators systemically administered the selective Hh inhibitor cyclopamine in ROP models and revealed that neovascularization was inhibited. Their results proved that Hh pathway activation has an important role in hypoxia-induced retinal neovascularization.

Liu et al.¹⁹ investigated the association between VEGF, retinal neovascularization, and the Hh signaling pathway in murine ROP models. Their results demonstrated that under normal oxygen concentrations, VEGF had low transcription and expression, high oxygen levels stimulated VEGF expression significantly, and cyclopamine inhibited VEGF expression by Hh blockade. The authors hypothesized that the Hh pathway participated in regulating VEGF levels in ROP and that blocking the Hh signaling pathway may be useful in the treatment of ROP.

The FZD4, LRP5, TSPAN12, and NDP genes are parts of the Wnt/b-catenin signaling pathway and have roles in cell survival, proliferation, and migration throughout the body. In the current study, an LRP5 mutation expected in FEVR was found in one patient. However, to our knowledge, this is the first reported case accompanied by anal atresia. Mutations in the LRP5, FZD4, and NDP gene have also been found in patients with ROP at varying frequencies.^20,21,22 In the current study, we diagnosed the patients as having FEVR based on the clinical presentation and medical data of the neonates, such as gestational age and birth weight. It is difficult to differentiate ROP and FEVR in premature infants. In our cases, the clinical findings mimicked ROP. However, there was no ridge appearance. Instead, all patients had flat neovascularizations in zone 2 and brush-paint-like hemorrhages. A new classification named ROPER (ROP vs. FEVR) was recently introduced due to the clinical resemblance of these two disorders.²³ John et al.²³ described ROPER in premature infants demonstrating retinal findings more typical of FEVR than ROP. They reported the differentiating fluorescein angiography features of ROPER as irregular buds of vascularization at the vascular/avascular junction, definite pruning of vessels, pinpoint hyperfluorescent areas, and segmental vascular leakage areas. Our patients did not undergo fluorescein angiography, but their retinal findings mimicked ROP. One patient had neovascularization on the iris. The differential diagnosis of these vascular disorders is important, because ROPER may have an unpredictable and long-term course.

In the course of organogenesis, cell numbers are regulated by balancing proliferation and cell death. During development, the Hh signaling pathway controls cell proliferation and survival.²⁴ Shh is a crucial survival factor for the neural stem cells, midbrain, forebrain, retina, and neural crest.^25,26 Furthermore, a recent study proved that Shh signaling functions in the maintenance of retinal endothelial tight junctions.¹¹

Retinal vessels have a high number of pericytes covering the endothelial cells. Signals from pericytes are essential for the formation and maintenance of the blood-retinal barrier.²⁷ Neovascular vessels are generally associated with disturbance of the blood-retinal barrier. The relationship between Shh signaling and vessel growth and barrier formation demonstrates the role of Shh in angiogenesis and barrier formation. A defect in the Shh pathway is likely to disrupt the blood-retinal barrier, impair oxygen supply to the retina, and cause retinal neovascularization.

HSCR is characterized by functional intestinal obstruction of a genetic etiology.²⁸ It involves abnormal development of the enteric nervous system resulting in intestinal dysfunction. Congenital anorectal malformations including anal atresia are common manifestations, with an incidence of 1 in 1,000-5,000 neonates.²⁹ The Hh family proteins (Shh, Ihh, Dhh) are secreted signaling molecules that manage the development and balancing of tissues.³⁰ Shh, Ihh, and Dhh are all expressed in the gut tube.³⁰ Gao et al.³¹ found that genetic polymorphisms in the Hh gene were associated with HSCR and anorectal malformations. Two of our patients were diagnosed with anal atresia and one was diagnosed with HSCR.

Study Limitations

The current study emphasizes the role of the Hh pathway in both retinal vascular and gut development and describes a new syndrome involving FEVR and GTMs. Pediatric surgeons should be aware of this association and refer neonates with GTMs to retina specialists.

Conclusion

In future investigations, analysis of clinical findings, retinal imaging with fluorescein angiography, and family genetic analysis may further our understanding of the genetics of neonatal vascular disease.

Ethics

Ethics Committee Approval: Obtained from the local ethics committee (KA19/306).

Informed Consent: Obtained from the parents of all patients.

Peer-review: Externally and internally peer reviewed.

Authorship Contributions

Concept: N.Ş.K., İ.A., A.T., Ö.K., Design: N.Ş.K., İ.A., A.T., Ö.K., Data Collection or Processing: N.Ş.K., İ.A., A.T., Ö.K., Analysis or Interpretation: N.Ş.K., İ.A., A.T., Ö.K., Literature Search: N.Ş.K., İ.A., A.T., Ö.K., Writing: N.Ş.K., İ.A., A.T., Ö.K.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.