|Year : 2019 | Volume
| Issue : 2 | Page : 171-178
Detection of FZD4, LRP5 and TSPAN12 genes variants in Malay premature babies with retinopathy of prematurity
Siti Zulaikha Nashwa Mohd Khair1
, Abdul Salim Ismail2, Zunaina Embong2, Abdul Aziz Mohamed Yusoff1
1 Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
2 Department of Ophthalmology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
|Date of Submission||11-Oct-2017|
|Date of Acceptance||01-Nov-2018|
|Date of Web Publication||19-Apr-2019|
Abdul Aziz Mohamed Yusoff
Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan
Source of Support: None, Conflict of Interest: None
Purpose: To determine the mutational analyses of familial exudative vitreoretinopathy (FEVR)-causing genes in Malay patients with retinopathy of prematurity (ROP) to obtain preliminary data for gene alterations in the Malay community.
Methods: A comparative cross-sectional study involving 86 Malay premature babies (ROP = 41 and non-ROP = 45) was performed from September 2012 to December 2014. Mutation analyses in (FEVR)-causing genes (NDP, FZD4, LRP5, and TSPAN12) were performed using DNA from premature babies using polymerase chain reaction (PCR) and direct sequencing. Sequencing results were confirmed with PCR-Restriction Fragment Length Polymorphism (RFLP).
Results: We found variants of FZD4, LRP5, and TSPAN12 in this study. One patient from each group showed a non-synonymous alteration in FZD4, c.502C>T (p.P168S). A synonymous variant of LRP5 [c.3357G>A (p.V1119V)] was found in 30 ROP and 28 non-ROP patients. Two variants of TSPAN12, c.765G>T (p.P255P) and c.*39C>T (3′UTR), were also recorded (29 and 21 in ROP, 33 and 26 in non-ROP, respectively). Gestational age and birth weight were found to be significantly associated with ROP (P value < 0.001 and 0.001, respectively).
Conclusion: Analysis of data obtained from the ROP Malay population will enhance our understanding of these FEVR-causing gene variants. The c.3357G>A (p.V1119V) variant of LRP5, and c.765G>T (p.P255P) and c.*39C>T variants of TSPAN12 could be common polymorphisms in the Malay ethnic group; however, this requires further elucidation. Future studies using larger groups and higher numbers of advanced cases are necessary to evaluate the relationship between FEVR-causing gene variants and the risk of ROP susceptibility in Malaysian infants.
Keywords: Genetic Variants; Malay; Premature Infant; Retinopathy of Prematurity
|How to cite this article:|
Mohd Khair SZ, Ismail AS, Embong Z, Mohamed Yusoff AA. Detection of FZD4, LRP5 and TSPAN12 genes variants in Malay premature babies with retinopathy of prematurity. J Ophthalmic Vis Res 2019;14:171-8
|How to cite this URL:|
Mohd Khair SZ, Ismail AS, Embong Z, Mohamed Yusoff AA. Detection of FZD4, LRP5 and TSPAN12 genes variants in Malay premature babies with retinopathy of prematurity. J Ophthalmic Vis Res [serial online] 2019 [cited 2020 Feb 21];14:171-8. Available from: http://www.jovr.org/text.asp?2019/14/2/171/256544
| Introduction|| |
Retinopathy of prematurity (ROP), previously called retrolental fibroplasia, is a vasoproliferative disease that is reported to be the leading cause of childhood blindness all over the world.,, This disease causes abnormal growth of retinal blood vessels, and is associated with multiple factors, including short gestational age, low birth weight, and extended oxygen exposure., In addition to environmental influences, genetic predisposition appears to be perhaps the strongest factor leading to ROP.,,,, Recently, researchers have shown increased interest in the genetic factors involved in ROP. It has been proposed that multiple alterations in the nuclear-encoded genes are responsible for development of ROP. Nevertheless, the precise mechanism conferring the genetic susceptibility to ROP is still being investigated.,,
ROP is considered to be part of familial exudative vitreoretinopathy (FEVR), a bilateral hereditary eye disorder, because of several clinical similarities between them., FEVR is well-known as a heterogeneous genetic disorder involving several genes and different genetic variations, including point mutations, deletions, or insertions. The genetic heterogeneity of FEVR cases has been documented by identification of causative genes, which include Norrie disease protein (
NDP), Frizzled-4 (FZD4), lipoprotein receptor-related protein 5 (LRP5), and Tetraspanin-12 (TSPAN12)., These genes all encode the ligand-receptor complex of Norrin/β-catenin signaling pathway, which is involved in the activation of vascularization in the retina., Due to the phenotypic similarity between FEVR and ROP, alterations in genes linked to FEVR are also considered to be associated with the development of ROP.
The Norrin-beta-catenin signaling pathway plays essential roles in c ontrolling retinal vascular development. Protein components of FZD4, LRP5 and a newly discovered component, TSPAN12 are expressed by endothelial cells in the eye, and this contributes to the formation of the Fzd4/Lrp5 complex. Binding of the Norrin ligand to the complex activates the downstream β-catenin signaling pathway, which consequently initiates expression of genes responsible for vascularization process in the retina. The pathway is simplified and illustrated in [Figure 1].,
|Figure 1. Norrin/-catenin signalling pathway. The binding of Norrin ligand to the receptor complex which consists of Fzd4, Lrp5 and Tspan12 protein components. The binding results in the activation of downstream -catenin signaling pathway which initiates the vascularisation process in the retina.,|
Click here to view
Most reports on FEVR-causing gene mutations are from western countries ,,,,, and Asian countries, especially from the Japan ,,,, and China ,,,,; however, there is no report of FEVR-causing gene mutations in Malaysian patients thus far. FZD4 and LRP5 variants were previously found to be in a range of 3% to 12% for overall ROP incidences., In the current study, we performed a comprehensive FEVR-causing gene (NDP, FZD4, LRP5, and TSPAN12)sequence analysis in 41 Malay premature babies with ROP.
| Methods|| |
A comparative cross-sectional study was conducted among Malay premature babies with birth weight ≤2000 g and gestational age at birth ≤36 weeks at the Hospital Universiti Sains Malaysia (HUSM) born between September 2012 to December 2014. The study adhered to the guidelines of the Declaration of Helsinki and was approved by the Universiti Sains Malaysia Ethics Committee. Informed consent was obtained from all parents or legal guardians prior to enrolment of the subjects for the study.
The premature babies were screened for ROP by the Pediatric Ophthalmology Team (and not the investigators) according to the ROP screening schedule. The first retinal examination of premature babies was performed depending on the risk factors for ROP, such as low birth weight (less than 1500 g), ventilator support for oxygen supplementation, sepsis, and intraventricular hemorrhage. For premature babies with risk factors for ROP (minimum of one risk factor), the first retinal examination was done at 32 weeks postmenstrual age (PMA). Whereas, for premature babies without risk factors for ROP, the first retinal examination was done at 34 weeks PMA.
If ROP was not detected during the first retinal examination (performed by the Pediatric Ophthalmology Team), the premature babies were re-examined every 2 weeks up to 38 weeks PMA. The next review was performed at 42 weeks PMA, and follow-ups were performed at the age of 6 months and 1-year-old. The premature babies were discharged from ROP screening if features of ROP were not detected at the age of 1 year. Upon confirmation of the absence of ROP in premature babies (at the age of 1 year), they were enrolled for the study as the non-ROP group and their blood samples were collected for genetic studies.
At any time of retinal examination, if ROP features were detected, the premature babies were enrolled for the study as the ROP group and their blood samples were collected for genetic studies. The premature babies were enrolled through consecutive sampling and were divided into ROP and non-ROP groups. The classification of ROP is based on clinical features described by the International Classification of ROP (ICROP). Diagnosis of ROP, at least in one eye, was required for enrollment of candidates in the ROP group. ROP was managed by the Pediatric Ophthalmology Team, and they assessed the development of ROP in premature babies based on its severity.
Whole blood samples were collected and extracted following the manufacturer's protocol using the QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). DNA concentration and purity were determined using the NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), this was followed by agarose gel electrophoresis using 1% gels for determining quality of DNA samples.
Polymerase Chain Reaction for NDP, FZD4, LRP5, and TSPAN12
Entire NDP, FZD4, LRP5, and TSPAN12 exons were amplified using sets of primers from previous reports.,, Polymerase chain reaction (PCR) was performed in a 20 μl reaction volume using the 2 U Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 200 μM of each dNTP, 10 μM of each primer, 70 ng of DNA template, and 4 μl of 5 × Phusion HF buffer (Thermo Fisher Scientific, Waltham, MA, USA).
Initial denaturation was performed at 98°C for 30 s, followed by 30 cycles of denaturation, annealing at respective temperatures (available in references) and extension at 72°C. Final extension time was five minutes. Gel electrophoresis using a 3% agarose gel was performed to analyze amplified fragments, the sizes of which were determined using 50 bp DNA marker. Favorable PCR products were purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced.
Direct Sequencing Analysis
Sequencing reactions were performed in both directions using forward and reverse primers. BigDye ® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) was used to sequence PCR products using an ABI Prism 3700 DNA Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA). Electropherogram results were aligned using NCBI BLAST (http://www.ncbi.nlm.nih.gov/blast). The complementary DNA (cDNA) sequences for FZD4 (NM_012193.3), LRP5 (NM_002335.3), TSPAN12 (NM_012338.3), and NDP (NM 000266.3) available in the database were used as references.
Validation of Variants Using PCR-RFLP
PCR-RFLP assays were used to validate the variants detected using DNA sequencing. Restriction enzymes used to cleave at detected single nucleotide polymorphism (SNP) sites were determined using several software applications available online, such as RFLP enzyme picker (http://www.insilicase.co.uk) and RestrictionMapper Version 3 (http://www.restrictionmapper.org). Variants were confirmed using FastDigest restriction enzymes (Thermo Fisher Scientific, Watham, MA, USA); c.502C>T confirmed using BseSI, c.3357G>A using XmiI, c.765G>T using MvaI, and c.*39C>T using BseNI.
Statistical data were analyzed using the Statistical Package for Social Science (SPSS) version 21 (IBM Corp., Armonk, N.Y., USA). The association of clinical factors and detected gene variants with ROP and non-ROP were tested using Chi-square or Fisher Exact test. P value of < 0.05 was considered as statistically significant.
| Results|| |
A total of 86 premature babies were enrolled (ROP = 41 and non-ROP = 45) for this study. Out of 86 premature babies, 49 (57.0%) were male and 37 (43.0%) were female. The group of premature babies diagnosed with ROP consisted of 22 (53.7%) males and 19 (46.3%) females, whereas, the non-ROP group consisted of 27 (60.0%) males and 18 (40.0%) females. There was no significant difference in number of candidates from either gender between the ROP and non-ROP groups (P = 0.553) [Table 1].
The mean gestational age at birth for the ROP group was 27.2 weeks (range, 24 to 34 weeks) whereas that for the non-ROP group was 30.2 weeks (range, 26 to 34 weeks). Gestational age at birth ≤28 weeks was significantly associated with ROP (P = <0.001) [Table 1].
Premature babies in the ROP group had birth weight ranging from 570 to 1500 g with a mean of 968 g. Whereas, non-ROP premature babies had a mean of 1396 g with the birth weight range lies between 700 to 2000 g. Birth weight <1000 g was significantly associated with ROP (P = 0.001) [Table 1].
According to staging for ROP, the highest number of cases were from stage three (n = 20, 48.8%), followed by those from stage two (n = 11, 26.8%), and stage one (n = 6, 14.6%) [Table 1].
A total of 86 premature babies were screened for genetic variants in FEVR-causing genes (NDP, FZD4, LRP5 and TSPAN12). Gene variants for FZD4, LRP5, and TSPAN12 were found upon screening. There was one heterozygous FZD4 gene variant in one patient from each group (ROP and non-ROP group) [Table 2]. The FZD4 c.502C>T (p.P168S, rs61735303) substitution was a partial non-synonymous change in exon 2 [Figure 2]a.
|Figure 2. Chromatograms show the variants detected in multiple genes which mutant was compared to the wild-type A. Base and protein position of variant detected in the exon 2 of FZD4 gene, c.502C>T (p.P168S) B. Base and protein position of variant detected in the exon 2 of LRP5 gene, c.3357G>A (p.V1119V) C. Base and protein position of variant detected in the exon 8 of TSPAN12 gene, c.765G>T (p.P255P).|
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One coding variant c.3357G>A (p.V1119V, rs556442) of the LRP5 gene in exon 15 was found [Figure 2]b. This synonymous substitution was found in 30 (73.2%) ROP and 28 (62.2%) non-ROP samples [Table 2].
Two variants of TSPAN12, c.765G>T (p.P255P, rs41623) and c.*39C>T (rs41622), were identified in both groups (ROP, 70.7% and 51.2%, respectively; non-ROP, 73.3% and 57.8%, respectively) [Figure 2]c and [Table 2]. Of 86 subjects, 47 (54.7%) were found to show both TSPAN12 gene variants.
There was no NDP variant found in this study. There was no significant difference for all gene variants between ROP and non-ROP groups.
| Discussion|| |
ROP is a multifactorial disease characterized by abnormal retinal vessel growth that can lead to blindness in infants. As the ROP phenotype resembles the FEVR phenotype, the four genes encoding the ligand-receptor complex of the Norrin/β-catenin signaling pathway were considered to be causative genes for the progression of ROP.,, The present study is the first to report mutational analyses of FEVR-causing genes in ROP patients from Malaysia. Previously, various types of mutations were found in the FZD4 gene such as point mutations, deletions, and insertions. However, sequencing of the entire FZD4 gene (translated region) revealed one type of heterozygous variant. The observed variant had a C to T transition at nucleotide position 502, which is responsible for alteration in the codon (CCC) coding for proline into codon (TCC) coding for amino acid serine, at amino acid position 168 (P168S). It was detected in only one patient in each group (ROP and non-ROP group). Double peaks were observed at the location that suggested the variant was transmitted in a heterozygous manner from the mother or father. This variant is possibly not pathogenic and does not contribute to the development of ROP in Malay infant patients. However, the non-synonymous or missense change might have knocked out the original function of the protein due to its occurrence at the functional site.
Previously, Ellset alreported the same change in the Canadian population and the occurrence of P168S was identified together with P33S. Six ROP and one control patient were found to have the coupled variants, whereas a single non-coupled change of P168S was observed in another control patient. These changes were classified as known polymorphisms with 1.42% allele frequency. In an earlier report in the population of Michigan, United States by Drenseret al, the same coupled changes were found in four patients with ROP and five with FEVR, and both were referred to as missense mutations as they were not found in control patients. Another study reported similar findings with a combination of changes in two of its Caucasian patients diagnosed with Coat's disease. In 2004, Toomes et al described the P168S alteration as a benign rare sequence variant where this missense change was found only once in 200 white control individuals (<0.3% frequency).
Proline is known to be an atypical amino acid, which plays a unique role in providing rigidity in protein conformations. Several studies have suggested that the proline to serine missense substitution might be a causative factor for FEVR or ROP, as protein rigidity was found to be compromised. The effect is more prominent with the involvement of another variant, P33S as this mutation is found in the sequence responsible for translocation signaling of the FZD4gene to plasma membrane., Thus, both the variants were considered to play a role in the development of ROP as reported by Boonstraet al. In a more recent study, Dailey et al revealed that the combination P33S/P168S change is the most prevalent FZD4 variant and is statistically significant to be associated with ROP and FEVR. The FZD4 coupling variants were considered to increase the incidence of retinal damaged causing ROP and intrauterine growth retardation, resulting in smaller newborns. The authors also suggested that these variants may be used as biomarkers for increased risk of development of ROP and preterm birth.
The LRP5 gene was previously reported to show various types of molecular abnormalities, such as missense or nonsense mutations, insertions, deletions, or splice site mutations in AD-FEVR and AR-FEVR patients. Kondo and colleagues analyzed the genetic variants of LRP5 in 53 Japanese patients with advanced ROP in 2013. Ten different single-base substitutions were discovered in seven patients, mostly in the coding sequence of the gene. We also identified a variant in exon 15 of the LRP5 gene, V1119V, which was known as a synonymous change or nonsense mutation due to no changes observed in protein sequence. According to Kondoet al, the synonymous change was classified by the NLHBI Exome Sequencing Project, Seattle, WA as common polymorphism with known minor allele frequency greater than 10%. In addition, Qinet al reported that it is a registered known polymorphism in the single nucleotide polymorphism database (dbSNP) and is categorized as a reference SNP. LRP5 is a receptor which functions along with FZD4 in the Norrin/β-catenin signaling pathway. The coding sequences of LRP5 are translated into a large number of amino acids, as much as 1615 individual proteins. Several studies have reported the presence of the c.3357G>A (p.V1119V) variant in FEVR and advanced ROP among Japanese and Chinese patients.,, It is not yet known whether this variant is involved in the progression of ROP.
Two variants of TSPAN12 in exon 8 were discovered in this study: a synonymous change c.765G>T (p.P255P, rs41623) and another–c.*39C>T in the 3′UTR. TSPAN12 is one of the components of tetraspanin which is known to be involved in various membrane-associated activities, such as cell proliferation and activation of signaling pathways. Its specific function is to allow the activation of the FZD4/LRP5 receptor complex by interacting with Norrin or LRP5. Complex formation of norrin/FZD4/LRP5 might be affected due to any defects in TSPAN12 which consequently affects vascularization. However, due to P255P which is a synonymous change, the relationship between the variant and ROP could not be further explained. P255P was also described by Kondoet al as non-pathogenic as it has a minor allele frequency of > 10%.
Both changes were discovered in more than half of the population in this study which includes 47 (54.7%) out of 86 subjects. The P255P change was found in all subjects with the 3′UTR variant. As proposed, the 3′UTR is involved in the regulatory pathways of transcription. The crucial function of the region is controlling gene expression as reported in previous studies involving SNPs at the 3′UTR., In addition, recent studies have proposed that any changes at the region are associated with susceptibility to diseases.,,,, Both variants were previously reported in Japanese and Indian populations as common single nucleotide polymorphisms and the details are available in dbSNP database (rs41622 and rs41623).,
In this study, we found no significant differences for all variants of FZD4, LRP5, and TSPAN12 when comparing the prevalence between both groups. The insignificant associations observed in our study between the said genes and ROP possibly due to a limited number of advanced cases enrolled in this study. This is postulated based on the results of a previous study performed in Japanese population involving severe stages of ROP patients which showed high prevalence of FZD4 or LRP5 variants in advanced ROP patients.
In this study, we did not detect any variants of the NDP gene in ROP patients. Possibly, this was due to the fact that ROP is a complex disease and is polygenetic; thus, the rate of detection of variants might be affected by the low frequency of NDP gene alterations in the population. In addition, samples obtained in the study were from the Malay ethnic group only and genotypic differences might be attributable to population stratification. There might be differences in the genetic predisposition between Malay ethnicity and populations from Western countries or Eastern Asia (Japan, China, and Korea).,, According to Ellset al, ROP has long been considered to be affected by racial factors. Researchers questioned the contribution of ethnicity to the development of ROP in the 1950s, when there were significant differences in the number of ROP cases among different races in a particular country.
Low gestational age leads to prematurity and low birth weight, which are two of the major causes of ROP., In this study, gestational age (P < 0.001) and birth weight (P = 0.001) were found to be significantly associated with ROP [Table 1]. These findings are in agreement with previous reports from Pakistan, Iran, Egypt, and Serbia, which showed a correlation between gestational age and birth weight with ROP.,,, The degree of retinal immaturity depends solely on the degree of prematurity, which supports the significant probable link between gestational age and ROP., Both, extreme prematurity (<28 weeks) and low birth weight (<1000 g) are proposed to have long term effects on visual functions that are believed to be significantly associated with ROP. A recent study suggested that babies with low birth weight tended to have narrower microvasculature development of the retinal arterioles.,
The major limitation of this study is the relatively small sample size and limited number of advanced/severe stage cases (stage four and five), which might influence the mutation rate detected. A previous study by Kondo and colleagues was used as a reference, wherein they reported that various types of mutations were detected in patients with advanced stages of ROP. However, gestational age and birth weight were the two factors associated with ROP despite the limited number of advanced cases used for this study. It is necessary to perform further studies using a larger sample size to enable the screening of FEVR-causing gene mutations in Malaysian ROP patients. Further studies might elucidate whether these gene mutations are risk factors for the development and progression of ROP for Malay Kelantan premature babies.
In conclusion, four variants of FEVR-causing genes were detected in our study involving Malay patients with ROP. They were identified as c.502C>T (p.P168S) of the FZD4 gene, c.3357G>A (p.V1119V) of the LRP5 gene, c.765G>T (p.P255P) and c.*39C>T (3′UTR) of the TSPAN12 gene. These variants were classified as known SNPs and their details are accessible in the dbSNP. Thus far, there is no compelling evidence to support that these polymorphisms play important roles in the development of ROP. However, it has been previously reported that patients with the non-synonymous variant of FZD4 have a significantly increased risk of developing ROP and of preterm birth. In addition to the molecular part, two of the major causes of ROP, low gestational age (prematurity) and low birth weight were found to be significantly associated with ROP. This is because degree of retinal immaturity depends on prematurity whereas low birth weight babies have narrower microvasculature development.
This work was supported by Short Term Grant (304/PPSP/61312022) and also Research University Grant (1001/PPSP/812110) from the Universiti Sains Malaysia.
Financial Support and Sponsorship
Short-term Grant (304/PPSP/61312022); and also Research University Grant (1001/PPSP/812110).
Conflicts of Interest
There are no conflicts of interest.
| References|| |
Hiraoka M, Takahashi H, Orimo H, Hiraoka M, Ogata T, Azuma N. Genetic screening of Wnt signaling factors in advanced retinopathy of prematurity. Mol Vis
Smith LE. Pathogenesis of retinopathy of prematurity. Growth Horm IGF Res
Eichenbaum JW, Mamelok A, Mittl RN, Orellana J. Treatment of Retinopathy of Prematurity. Chicago, IL: Year Book-Mosby; 1991.
Bizzarro MJ, Hussain N, Jonsson B, Feng R, Ment LR, Gruen JR, et al. Genetic susceptibility to retinopathy of prematurity. Pediatrics
Kondo H, Kusaka S, Yoshinaga A, Uchio E, Tawara A, Tahira T. Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity. Mol Vis
Holmström G, Van Wijngaarden P, Coster DJ, Williams KA. Genetic susceptibility to retinopathy of prematurity: The evidence from clinical and experimental animal studies. Br J Ophthalmol
Shastry BS. Genetic Susceptibility to advanced retinopathy of prematurity (ROP). J Biomed Sci
Clevers H. Eyeing up new Wnt pathway players. Cell
Ye X, Wang Y, Nathans J. The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease. Trends Mol Med
Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K, Breakefield XO, et al. A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat Genet
Robitaille J, MacDonald ML, Kaykas A, Sheldahl LC, Zeisler J, Dubé MP, et al. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet
Toomes C, Bottomley HM, Jackson RM, Towns KV, Scott S, Mackey DA, et al. Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q. Am J Hum Genet
Poulter JA, Ali M, Gilmour DF, Rice A, Kondo H, Hayashi K, et al. Mutations in TSPAN12 cause autosomal-dominant familial exudative vitreoretinopathy. Am J Hum Genet
Nikopoulos K, Gilissen C, Hoischen A, van Nouhuys CE, Boonstra FN, Blokland EA, et al. Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy. Am J Hum Genet
Pefkianaki M, Hasanreisoglu M, Suchy SF, Shields CL. Familial Exudative Vitreoretinopathy with a Novel LRP5 Mutation. J Pediatr Ophthalmol Strabismus
Kondo H, Hayashi H, Oshima K, Tahira T, Hayashi K. Frizzled 4 Gene (FZD4) Mutations in patients with familial exudative vitreoretinopathy with variable expressivity. Br J Ophthalmol
Kondo H, Qin M, Kusaka S, Tahira T, Hasebe H, Hayashi H, et al. Novel mutations in Norrie disease gene in Japanese patients with Norrie disease and familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci
Kondo H, Kusaka S, Yoshinaga A, Uchio E, Tawara A, Hayashi K, et al. Mutations in the TSPAN12 gene in Japanese patients with familial exudative vitreoretinopathy. Am J Ophthalmol
Xu Y, Huang L, Li J, Zhang Q, Fei P, Zhu X, et al. Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy. Mol Vis
Fei P, Zhang Q, Huang L, Xu Y, Zhu X, Tai Z, et al. Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy. Mol Vis
Yang H, Li S, Xiao X, Wang P, Guo X, Zhang Q. Identification of FZD4 and LRP5 mutations in 11 of 49 families with familial exudative vitreoretinopathy. Mol Vis
Yang H, Li S, Xiao X, Guo X, Zhang Q. Screening for NDP mutations in 44 unrelated patients with familial exudative vitreoretinopathy or Norrie disease. Curr Eye Res
Yang H, Xiao X, Li S, Mai G, Zhang Q. Novel TSPAN12 mutations in patients with familial exudative vitreoretinopathy and their associated phenotypes. Mol Vis
Corpus K, Jimenez JM, Anzures R, Bascuna RI, Ventura R, Reandelar M, et al. Proposed new retinopathy of prematurity screening criteria: Evidence for including older and heavier Filipino premature babies. Philipp J Ophthalmol
Toomes C, Downey L. Familial exudative vitreoretinopathy, autosomal dominant. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJ, et al. editors. GeneReviews®2005. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1147/
. [Last accessed on 2016 Jul 15].
Ells A, Guernsey DL, Wallace K, Zheng B, Vincer M, Allen A, et al. Severe retinopathy of prematurity associated with FZD4 mutations. Ophthalmic Genet
Drenser KA, Dailey W, Vinekar A, Dalal K, Capone A, Trese MT. Clinical presentation and genetic correlation of patients with mutations affecting the FZD4 gene. Arch Ophthalmol
Toomes C, Bottomley HM, Scott S, Mackey DA, Craig JE, Appukuttan B, et al. Spectrum and frequency of FZD4 mutations in familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci
Nallathambi J, Shukla D, Rajendran A, Namperumalsamy P, Muthulakshmi R, Sundaresan P. Identification of novel FZD4 mutations in Indian patients with familial exudative vitreoretinopathy. Mol Vis
Boonstra FN, Van Nouhuys CE, Schuil J, de Wijs IJ, van der Donk KP, Nikopoulos K, et al. Clinical and molecular evaluation of probands and family members with familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci
Dailey WA, Gryc W, Garg PG, Drenser KA. Frizzled-4 Variations associated with retinopathy and intrauterine growth retardation: A potential marker for prematurity and retinopathy. Ophthalmology
Qin M, Hayashi H, Oshima K, Tahira T, Hayashi K, Kondo H. Complexity of the genotype–phenotype correlation in familial exudative vitreoretinopathy with mutations in the LRP5 and/or FZD4 genes. Hum Mutat
Kondo H. Complex genetics of familial exudative vitreoretinopathy and related pediatric retinal detachments. Taiwan J Ophthalmol
Garcia-Espana A, Chung PJ, Sarkar IN, Stiner E, Sun TT, Desalle R. Appearance of new tetraspanin genes during vertebrate evolution. Genomics
Junge HJ, Yang S, Burton JB, Paes K, Shu X, French DM, et al. TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt induced FZD4/beta-catenin signaling. Cell
Zhu Q, Chen Y, Dai J, Wang B, Liu M, Wang Y, et al. Methylenetetrahydrofolate reductase polymorphisms at 3'-untranslated region are associated with susceptibility to preterm birth. Translational Pediatrics
Hino K, Sato H, Sugai A, Kato M, Yoneda M, Kai C. Downregulation of Nipah virus N
mRNA occurs through interaction between its 3' untranslated region and hnRNP D. J Virol
Melanson BD, Cabrita MA, Bose R, Hamill JD, Pan E, Brochu C, et al. A novel cisacting element from the 3'UTR of DNA damage binding protein 2 mRNA links transcriptional and posttranscriptional regulation of gene expression. Nucleic Acids Res
Adams LA, Möller M, Nebel A, Schreiber S, Van Der Merwe L, Van Helden PD, et al. Polymorphisms in MC3R promoter and CTSZ 3'UTR are associated with tuberculosis susceptibility. Eur J Hum Genet
Akhter Q, Masood A, Ashraf R, Majid S, Rasool S, Khan T, et al. Polymorphisms in the 3'UTR of the human leptin gene and their role in hypertension. Mol Med Rep
Chen Z, Brant SR, Li C, Shrestha UK, Jiang T, Zhou F, et al. CTLA4 -1661A/G and 3'UTR long repeat polymorphisms are associated with ulcerative colitis and influence CTLA4 mRNA and protein expression. Genes Immun
Pan ZW, Luo CF, Liu ZJ, Li JC. RET 3'UTR polymorphisms and its protective role in Hirschsprung disease in southeastern Chinese. J Pediatr Surg
Silva ID, Muniz YC, Sousa MC, Silva KR, Castelli EC, José Filho CG, et al. HLA-G 3'UTR polymorphisms in high grade and invasive cervico-vaginal cancer. Hum Immunol
Kaur I, Musada G, Jalali S, Kekunnaya R, Gaddam P, Chakrabarti S. Genetic Screening of TSPAN12, NDP and FZD4 Genes in Indian Patients with Retinopathy of Prematurity. Invest Ophthalmol Vis Sci
Zunaina E, Abdul-Salim I, Siti-Zulaikha-Nashwa MK, Mohamed-Yusoff AA, Ramli N, Ibrahim MI, et al. Malay premature infants with retinopathy of prematurity: Risk factors and screening of NDP gene mutation. Journal of Biomedical and Clinical Sciences (JBCS)
Darlow BA, Hutchinson JL, Henderson-Smart DJ, Donoghue DA, Simpson JM, Evans NJ. Prenatal risk factors for severe retinopathy of prematurity among very preterm infants of the Australian and New Zealand Neonatal Network. Pediatrics
Hellström A, Smith LE, Dammann O. Retinopathy of prematurity. Lancet
Knežević S, Stojanović N, Oros A, Savić D, Simović A, Knežević J. Analysis of risk factors in the development of retinopathy of prematurity. Srpski arhiv za celokupno lekarstvo
Hakeem AH, Mohamed GB, Othman MF. Retinopathy of prematurity: A study of prevalence and risk factors. Middle East Afr J Ophthalmol
Sohaila A, Tikmani SS, Khan IA, Atiq H, Akhtar ASM, Kumar P, et al. Frequency of Retinopathy of Prematurity in Premature Neonates with a Birth Weight below 1500 Grams and a Gestational Age Less than 32 Weeks: A Study from a Tertiary Care Hospital in a Lower-Middle Income Country. PloS One
Alizadeh Y, Zarkesh M, Moghadam RS, Esfandiarpour B, Behboudi H, Karambin MM, et al. Incidence and risk factors for retinopathy of prematurity in North of Iran. J Ophthalmic Vis Res
Lermann VL, Fortes Filho JB, Procianoy RS. The prevalence of retinopathy of prematurity in very low birth weight newborn infants. Jornal De Pediatria
Fierson WM, Saunders RA, Good W, Palmer EA, Phelps D, Reynolds J, et al. Screening examination of premature infants for retinopathy of prematurity. Pediatrics
Mitchell P, Liew G, Rochtchina E, Wang JJ, Robaei D, Cheung N, et al. Evidence of arteriolar narrowing in low-birth-weight children. Circulation
Sun C, Ponsonby AL, Wong TY, Brown SA, Kearns LS, Cochrane J, et al. Effect of Birth Parameters on Retinal Vascular Caliber The Twins Eye Study in Tasmania. Hypertension
[Figure 1], [Figure 2]
[Table 1], [Table 2]