Severe ROP can lead to blindness in preterm infants, which is characterized by abnormal retinal vessel growth. FZD4, LRP5, and ZNF408 are involved in activation of the Norrin/β-catenin signaling pathway and are considered the causative genes for ROP progression, similar to that for FEVR.[4, 8, 19, 20] This study first reported a mutation analysis of the FEVR pathogenic gene in Chinese ROP patients. Various types of mutations in the NDP gene have been reported.[21] However, in this study, entire NDP gene sequencing (translation region) revealed a heterozygous variant comprising a transition from T to C at nucleotide position 715, and only two patients in each group were identified as having this alteration. This variant might not be pathogenic and might not be involved in the development of ROP.
As is known, proline is an atypical amino acid that plays a unique role in the rigidity of protein conformations. Several studies found that proline to serine missense substitutions cause compromised protein rigidity, indicated that it might be a causative factor for FEVR or ROP.[22, 23] FZD4 coupling variants might increase the incidence of retinal damage, which can lead to ROP. It was also indicated that biomarkers for an increased risk of ROP development might comprise these variants.[24]
It has been previously reported that LRP5 in FEVR patients is associated with various types of molecular abnormalities. The genetic variants of LRP5 with 10 different single‑base substitutions were analyzed in 53 Japanese patients with advanced ROP in 2013, and variants in the coding sequence of this region were mostly discovered.[4] We also identified a variant in exon 2, exon 9, exon 10, and exon 19 of LRP5 (Q89R, D666N, E644E, and D1363D, which were determined to be nonsynonymous, synonymous, or splicing change mutations based on the unchanged protein sequence. The NLHBI GO Exome Sequencing Project (ESP, Seattle, WA, USA) was used to classify synonymous changes as common polymorphisms, with minor allele frequencies known to be less than 10%.[4] In the Norrin/β-catenin signaling pathway, LRP5 is a receptor that functions with FZD4.[25] The presence of the c.3357G > A (p.V1119V) variant was reported previously in severe ROP in a Chinese and Japanese population.[21, 26] These variants might be associated with the development of ROP.
Variants of TSPAN12 in the 3ʹUTR and exon 3 were identified in this study, including a splicing change, c.67-9delT (rs774111149), and another c.*1139A>T (rs192303288) variant in the 3ʹUTR. TSPAN12 is one component of a four-tetraspanin protein and is involved in various activities associated with cell proliferation and signaling pathways;[27] its specific function is to activate the FZD4/LRP5 receptor complex by interacting with Norrin or LRP5.[28] Any defects in TSPAN12 might affect formation of the Norrin/FZD4/LRP5 complex, thereby affecting blood vessel formation.
Both changes were discovered in one tenth of this study population (13 of 134 subjects). The c.*1139A > T (rs192303288) change was found in all subjects with the 3ʹUTR variant. As proposed, the 3ʹUTR is involved in regulatory pathways of transcription. In all subjects with the 3ʹUTR variant, the c.*1139A > T (rs192303288) change was found.[29] As reported in previous studies, the role of this region is to control gene expression involving SNPs at the 3ʹUTR.[30] Furthermore, some studies showed that any 3ʹUTR variant might be associated with susceptibility to some diseases.[31]
Novel mutations in ZNF408 were found in this study. ZNF408 encodes a transcription factor of the zinc finger family. Three variants of ZNF408 in downstream bases, exon 4, and exon 5 were discovered as follows: the unknown change rs146198477, non-frameshift change c.581_592del AGTGGTG (p. Val194_Val197del AVVTEinsA, rs148055528), and nonsynonymous change c.1007C > T (p. S336L, rs150368802) in the exon. According to the type and number of zinc fingers, zinc finger genes can be separated into many subclasses.[32] Each finger constitutes an independent domain, which is stabilized by zinc ions attached to cysteine and histidine, along with a hydrophobic core in the inner structure. Some reports suggested that variants of ZNF408 lead to abnormal retinal vascularization in humans and might be related to disease processes in FEVR.[33] However, the molecular mechanism of ZNF408-associated ROP is not well known. Our report suggests that the p.S336L mutation disrupts the expression of important genes in angiogenesis and sheds further light on the molecular mechanisms underlying ZNF408-associated ROP.
There were some limitations to the present study. We only did gene polymorphisms analysis for type 1 ROP or threshold ROP patients, which may lead to visual impairment. Most of Type 2 or mild ROP recovered spontaneously, and major parents of these infant declined to participant this study. It could be necessary to conduct further studies with larger sample sizes to screen Norrin signaling gene mutations in Chinese ROP patients and to clarify whether these mutations are risk factors for the occurrence and development of ROP. Further, several infant characteristics varied between severe ROP and non-ROP groups in this study.
In conclusion, five Norrin signaling genes involved in ROP were detected in the present study based on a Chinese population. They were identified as c.*3770A > T (rs182049165) and c.*2971T > C (rs10898563) of the FZD4 gene, c.575 + 6T > C (rs4988322) of the LRP5 gene, c.*715T > C (rs3747350) of the NDP gene, c.67-9delT(rs774111149) of the TSPAN12 gene, and novel founder mutations c.1007C > T (p.S336L) and c.581_592del AGTGGTGACAGA (p. Val194_Val197del AVVTEinsA, rs148055528) of the ZNF408 gene. These mutations were classified into SNPs, for which details are available in the dbSNP. The present study suggested that patients with the non-synonymous variant in FZD4, the splicing variant of LRP5, and non-frameshift variant of ZNF408 might have a significantly increased risk for the development ROP.