Molecular Vision 2021; 27:632-642 <http://www.molvis.org/molvis/v27/632>
Received 25 June 2021 | Accepted 18 November 2021 | Published 20 November 2021

Five novel copy number variations detected in patients with familial exudative vitreoretinopathy

Jia Luo,1 Jing Li,1 Xiang Zhang,1 Jia-Kai Li,1 Hao-Jie Chen,2 Pei-Quan Zhao,1 Ping Fei1

1Department of Ophthalmology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2Department of Urology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

Correspondence to: Ping Fei, Department of Ophthalmology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China; Phone: 86-13636342098; FAX: 86-21-65795173; email: feiping@xinhuamed.com.cn

Abstract

Purpose: Familial exudative vitreoretinopathy (FEVR) is an inherited retinal vascular disease genetically heterogeneous with multiple causative genes. The aim of this study is to report five novel copy number variation (CNV) regions in FEVR patients and to investigate the possible contributions of novel CNVs to FEVR.

Methods: In this study, 824 FEVR families were collected. All cases were performed using the targeted next generation sequencing (NGS) assay, and families with no definite pathogenic mutations in FEVR genes were screened for CNVs according to the NGS results. Droplet digital polymerase chain reaction (ddPCR) testing was introduced to validate the screened CNV regions. We also reviewed the clinical presentations of the probands and affected family members associated with the novel CNVs and conducted segregation analysis.

Results: Five CNVs in five patients were detected in this study: heterozygous deletions of kinesin family member 11 (KIF11) exons 2–4, KIF11 exon 11, KIF11 exons 1–10, tetraspanin-12 (TSPAN12) exons 1–3, and low-density lipoprotein receptor-related protein 5 (LRP5) exons 19–21. Among the five affected families, TSPAN12 exons 1–3 heterozygous deletion and LRP5 exons 19–21 heterozygous deletion originate from the mother and the father of the proband, respectively. No other family members manifested as FEVR except for the probands. The correlation between disease severity and CNV loci seems uncertain.

Conclusions: Five novel CNV loci in FEVR patients were uncovered in this study, including one maternally-inherited and one paternally-inherited CNV region. Though there is no evidence of co-segregation between these CNVs and FEVR, our findings suggest novel genetic risk factors for FEVR.

Introduction

Familial exudative vitreoretinopathy (FEVR) is an inherited vitreoretinal disorder first described in 1969 [1]. As a retinal vascular disease, FEVR is characterized by avascular zones or incomplete vascularization of the peripheral retina, and the clinical presentations of FEVR vary in different people from mild peripheral avascularity to severe retinal detachment (RD) [2-4]. Generally, FEVR is associated with mutations in genes involved in the Wnt/Norrin signaling pathway, including genes encoding the low-density lipoprotein receptor-related protein 5 (LRP5), Norrie disease protein (NDP), tetraspanin-12 (TSPAN12), and the receptor frizzled-4 (FZD4). Mutations in zinc finger protein 408 (ZNF408) and kinesin family member 11 (KIF11) were also identified as genetic causes for FEVR via other mechanisms [5-9]. The mutations mentioned above can only explain the causation in approximately 40% to 50% of FEVR patients, and the remaining genetic causes are still unknown [10,11].

Copy number variation (CNV), a form of chromosome submicroscopic structural variation, is defined as the deletion or duplication of a DNA segment whose size is more than 1 kb [12,13]. Studies have suggested that CNVs contribute to many human disorders [14-16] through several mechanisms: altering gene dosage, changing the 3D architecture of the genome, forming chimeric genes, and so on [17-19]. Many novel CNVs have recently been found in ophthalmic diseases, including retinitis pigmentosa [20], inherited retinal degeneration [21,22], and esotropia [23]. Previous genetic studies on FEVR focused on mutations in nucleic acids known as single nucleotide variants (SNV), while the possible role of CNV in FEVR has rarely been investigated [24-26]. Here, we identified five novel CNV regions in FEVR patients, which may contribute to the diagnosis of FEVR.

Methods

Subjects

This study was approved by the Institutional Research Committee of Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, and conducted under the light of the Declaration of Helsinki. We collected 824 patients who were clinically diagnosed with FEVR in the ophthalmology department of Xinhua Hospital between April 2015 and July 2019. Patients with a history of premature birth and oxygen inhalation were excluded. Written informed consent was obtained from the parents or guardians of the patients before the experiments were conducted.

Diagnosis

The FEVR diagnosis was made based on medical recordings and ophthalmic examinations, including slit-lamp biomicroscopy, B-scan ultrasound, RetCam III (Clarity Medical Systems, Pleasanton, CA), Optos 200Tx (Optos Inc., Marlborough, MA), and fundus fluorescein angiography (FFA). According to the examinations, patients presenting with at least one of the following typical clinical features were diagnosed as having FEVR: RD, retinal folds, vitreous hemorrhage, retinal neovascularization, peripheral avascular zones, severe subretinal exudates, or vitreoretinal dragging with macular ectopia [27]. All probands and their family members were examined and staged for FEVR, as described previously (Appendix 1) [28,29].

CNV screening and validation

Blood samples of probands and their family members were collected and used to extract genomic DNA using Gentra PureGene blood kits (Qiagen, Valencia, CA). The retinal disease panel used in this study involved 463 targeted genes (Appendix 2), and all enrolled cases underwent panel-based targeted next generation sequencing (NGS) assay. The standard Illumina libraries were prepared using a DNA Sample Prep Reagent Set (MyGenostics Inc., Beijing, China), and a GenCap capture kit (MyGenostics Inc., Beijing, China) was used to capture the regions containing the 463 targeted genes. Extracted DNA was quantified using Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA) and sheared into DNA fragments via Diangenode Bioruptor® Plus. Captured fragments were removed from the solution using streptavidin-coated magnetic beads (Dynabeads® MyOne™ Streptavidin T1, Thermo Fisher Scientific) and subsequently eluted. DNA fragments were enriched and sequenced on an Illumina HiSeq X ten sequencer for paired-reading of 150 bp. After interpreting the results of the Illumina HiSeq X ten sequencer into reads, a Burrows-Wheeler Aligner (BWA; ver. 0.7.11) was used for alignment between the clean reads and the human reference genome (hg19) [30]. Raw variants were analyzed via the genome analysis toolkit (GATK) Haplotype Caller and annotated with ANNOVAR software (http://annovar.openbioinformatics.org/en/latest/) according to the following databases: 1000 Genome Project, Exome Sequencing Project, Exome Sequencing Project, and the Human Gene Mutation Database (HGMD) [31]. Patients with no definite pathogenic mutations in FEVR genes were screened for potential CNVs by CapCNV analysis through CNVkit software, and the pathogenicity of the CNVs was classified into five categories, “pathogenic,” “likely pathogenic,” “uncertain significance,” “likely benign,” and “benign,” according to the American College of Medical Genetics and Genomics (ACMG) guidelines [32,33].

Then, ddPCR was performed to validate the CNV regions in the probands according to a previous study [34]. The genomic DNA was digested using an enzymatic digestion mixture at 37 °C for 1 h, followed by inactivation at 65 °C for 15 min. After DNA digestion, template DNA at a concentration of 40 ng/µL was used for ddPCR analysis performed on a QX100 system (BioRad Laboratories, Inc., Shanghai, China). PCR amplifications were conducted three times for each sample, with an optimized PCR thermal profile. Primers were designed by Primer Premier 5.0, and the sequences of primers are displayed below (Appendix 3). QuantaSoft v.1.2.10.0 software (BioRad Laboratories, Inc., Shanghai, China) was used to analyze the results.

Statistical analysis

The clinical characteristics and ophthalmological findings of the probands and affected family members were recorded for genotype–phenotype correlation analysis. Descriptive statistics were presented as median and range, statistical data were analyzed by the statistical analysis system (version 9.4), and a p value less than 0.05 was accepted as statistically significant.

Results

In this study, 824 unrelated FEVR families were enrolled, and 406 families without definite pathogenic mutations in FEVR genes were screened for CNVs. Five novel CNV loci were confirmed in five probands, one female and four males; the median age at diagnosis was 5 years (range: 4 months to 7 years of age). The demographic and clinical data are summarized in Table 1. The age at diagnosis was significantly different between inherited CNV probands and de novo CNV probands (p = 0.0056 < 0.05).

Five novel CNVs were detected

Among the 406 probands with no definite pathogenic mutations in FEVR genes, eight different CNV regions were screened in eight unrelated patients by NGS testing, including one duplication and seven heterozygous deletions. KIF11 accounted for three CNV loci (3/8, 37.5%), followed by TSPAN12 (2/8, 25%), LRP5 (1/8, 12.5%), FZD4 (1/8, 12.5%) and COL11A1 (1/8, 12.5%). Among them, heterozygous deletion of exon 1 of FZD4 and the whole gene of TSPAN12 have been reported previously [25,26]. There were two CNV regions involved in the whole gene, one is TSPAN12 heterozygous deletion, and the other is the duplication of COL11A1. Detailed information about the testing results is listed in Table 2. Notably, mutations were detected in two patients. Two heterozygous mutations were detected in patient No. 7, which are located at chr10–58232699, involving KIF11, and chr17–58232699, involving CA4, both resulting in DNA and protein changes. Both were predicted as mutations of uncertain pathogenicity, according to the ACMG. Patient No. 3 had a hemizygous mutation located at ChrX-43808330, containing the NDP gene, leading to a DNA mutation whose pathogenicity is uncertain based on the ACMG.

A ddPCR assay was performed in 24 cases from the eight families described above. Following the ddPCR assay, five of the eight CNV regions harboring FEVR-associated genes were validated in seven cases, five probands and two of their family members: KIF11 exons 2–4 heterozygous deletion, TSPAN12 exons 1–3 heterozygous deletion, LRP5 exons 19–21 heterozygous deletion, KIF11 exon 11 heterozygous deletion, and KIF11 exons 1–10 heterozygous deletion. The results of the ddPCR are shown in Figure 1. According to validations of CNVs in family members, TSPAN12 exons 1–3 heterozygous deletion was confirmed to be maternally inherited, and LRP5 exons 19–21 heterozygous deletion was paternally inherited, while three CNVs in KIF11 were de novo. The results are shown in Figure 2.

Phenotypes of FEVR patients and affected family members

The clinical manifestations of the five probands are summarized in Table 1, and fundus photography is shown in Figure 3. KIF11 exons 2–4 heterozygous deletion was detected in a 6-year-old boy (patient No. 1). The fundus showed vascular and macular dragging from the optic disc to the temporal retina in bilateral eyes, and apparent macular ectopia was found in the left eye (Figure 3A). A 7-year-old girl (patient No. 2) was identified as carrying a KIF11 exons 1–10 heterozygous deletion; fundus photography revealed a retinal fold extending to the temporal retina, with little exudates around the fold and mild vascular abnormalities in the right eye. Her left eye showed mild dragging from the optic disc to the temporal retina and was accompanied by retinal pigment epithelium atrophy (Figure 3B). One novel CNV of the KIF11 exon 11 heterozygous deletion was found in a 5-year-old boy (patient No. 3). He presented with posterior synechia of the iris and a falciform retinal fold in the right eye. In his left eye, the fundus was invisible with a flat anterior chamber and pupil occlusion; B-scan ultrasound indicated total retinal detachment (Figure 3C). The parents of these three probands underwent FFA examination, and none of them showed FEVR features.

Patient No. 4, a 9-month-old boy with a heterozygous deletion of TSPAN12 exons 1–3, presented with vitreoretinal traction in both eyes, which resulted in a radial retinal fold in the right eye and a dragged disc with ectopic macular in the left eye (Figure 3D). His mother was confirmed to carry the same CNV but with no clinical phenotypes of FEVR, according to fundus examinations. A heterozygous CNV of LRP5 exons 19–21 deletion was found in a 4-month-old boy (patient No. 5). Corneal opacity, shallow anterior chamber, and total retinal detachment were present in his right eye, according to RetCam and B-scan ultrasound examinations. Compared to the right eye, his left eye was milder, with peripheral nonperfusion area and neovascularization in the retina (Figure 3E). His father, who carried the same CNV, showed no features of FEVR on fundus examinations.

Discussion

FEVR is a genetic ophthalmic disease with various inheritance modes and phenotypes. Up to now, at least eight genes have been confirmed as FEVR-associated, disease-causing genes: FZD4, TSPAN12, NDP, LRP5, ZNF408, KIF11, CTNNB1, and JAG1 [9,35-42]. Most studies focus on the point mutations in these genes leading to FEVR, while few studies investigate the role of CNV, a pattern of DNA structural variants, in FEVR [24-26]. CNVs are deletions or duplications of DNA segments that can influence at least five times more variable base pairs than SNVs and lead to a greater impact on disease phenotypes [43].

Herein, we detected five novel CNV regions in five out of 406 FEVR patients without definite pathogenic mutations in FEVR genes. Three of them affect the exons of KIF11, including exons 2–4, exon 11, and exons 1–10. Previous studies demonstrated that patients with mutations in KIF11 present with microcephaly, mental retardation, lymphedema, and retinopathy with lacunar chorioretinal atrophic lesions [44], while other studies indicated that whether FEVR is associated with the syndrome caused by KIF11 mutations remains uncertain [45,46]. In our study, no patients harboring CNVs corresponding to KIF11 manifested as microcephaly, mental retardation, or lymphedema. A novel heterozygous CNV of LRP5 exons 19–21 deletion was detected in patient No. 5. Mutations in LRP5 often contribute to orthopedic diseases, such as osteoporosis or high bone mass [47,48]. However, the proband and his father, who harbored CNV in LRP5, showed normal bone mineral density and had no orthopedic history. Studies have reported that clinical presentations in patients with large deletions are not more severe than those in a patient with point mutation [26]. Therefore, we suggest that absent or undiagnosed complications of the extraocular system may be partially due to the different patterns of mutations.

Ocular manifestations of the ten eyes studied were diversified from peripheral retinal nonperfusion to total retinal detachment. All eyes were staged for FEVR. Except for one eye that was classified as a stage of 1B, the remaining eyes were all staged from 3A to 5B, indicating a severe fundus condition. Previous studies have demonstrated that the severity of FEVR is associated with the gene loci in patients with SNVs [11,49]. In this study, there was no significant relationship between the severity of FEVR and gene loci in the five probands, which may be due to the different mutation types and the limited population in our study. The contralateral eyes of patient No. 5 were staged for 5B and 1B, respectively, and the asymmetric ocular presentation was described as a characteristic of FEVR previously [50].

According to ddPCR verification results, three CNVs corresponding to KIF11 are de novo variations, and the heterozygous deletion of TSPAN12 exons 1–3 detected in patient No. 4 originated from the mother; the heterozygous deletion of LRP5 exons 19–21 identified in patient No. 5 is inherited from the father. Notably, among the inherited-CNV families, neither the mother harboring a TSPAN12 exons 1–3 heterozygous deletion nor the father carrying an LRP5 exons 19–21 heterozygous deletion had typical clinical manifestations of FEVR, according to fundus photography results, which may be consistent with the fact that the severity of FEVR varies among family members [51]. A previous study suggested that, except for the pathogenic variant, additional disease modifiers can influence penetrance [52]. DNA methylation may act as an epigenetic modification that contributes to incomplete penetrance in patients with known mutations [53,54]. Herein, the phenomenon of inconsistency between genotype and phenotype may be explained as incomplete penetrance, which is associated with many other mechanisms, such as DNA methylation. Further studies should be conducted to confirm this detailed mechanism. In addition, the significant difference (p = 0.0056 < 0.05) between the diagnostic age of the probands with inherited CNVs and that of the probands with de novo CNVs implied that the inherited CNVs may predispose patients to earlier occurrences of FEVR.

In our study, patient No. 3, who harbored a de novo CNV of KIF11 exon 11 deletion, was found to have a hemizygous mutation corresponding to the NDP gene, which the ACMG predicted to have unknown pathogenicity. Whether the CNV or the SNV is the causal variation of FEVR is inconclusive. This mutation was detected in patient No. 3’s mother, who showed no FEVR clinical features, contradicting the hypothesis that SNV causes the FEVR phenotype. A study demonstrated that the impact on the phenotype triggered by CNV is generally considered stronger than SNV [26]. Additionally, all five CNVs detected in this study were classified as “pathogenic,” according to ACMG guidelines. Taken together, our results support the idea that the deletion of KIF11 exon 11 is likely to be a causal variation in the clinical manifestations of FEVR.

Our study has several limitations. First, we did not validate the expression quantity of these novel CNVs in normal controls, which reduced our ability to evaluate the significance of the novel CNVs. Second, the fact that the cases were diagnosed and referred to the ophthalmology department of a tertiary health care center may represent referral bias. Nevertheless, we collected a population of 824 FEVR families and detected five novel CNVs.

In conclusion, CNV is known to be a genetic risk factor for many diseases. Herein, we uncovered five novel CNVs in FEVR patients and hypothesized that they are genetic risk factors for the occurrence of FEVR. To investigate their determined significance, more experiments are warranted.

Appendix 1. Stage definition of FEVR.

Appendix 2. 463 targeted genes tested by NGS.

Appendix 3. Sequences of primers and probes used for ddPCR assay, GAPDH is served as internal reference.

Acknowledgments

The authors thank all the patients and their family members for their participation. This research was supported by Grant 81770963, 81770964 from National Natural Science Foundation of China and Grant 2019YFA0800801 from National Key Research and Development Program of China. We declare that no author has reported a financial conflict of interest in this manuscript. Pei-Quan Zhao (zhaopeiquan@xinhuamed.com.cn) and Ping Fei (feiping@xinhuamed.com.cn) are both the co-corresponding authors to this study.

References

  1. Criswick VG, Schepens CL. Familial exudative vitreoretinopathy. Am J Ophthalmol. 1969; 68:578-94. [PMID: 5394449]
  2. Miyakubo H, Hashimoto K, Miyakubo S. Retinal vascular pattern in familial exudative vitreoretinopathy. Ophthalmology. 1984; 91:1524-30. [PMID: 6084219]
  3. Boonstra FN, van Nouhuys CE, Schuil J, de Wijs IJ, van der Donk KP, Nikopoulos K, Mukhopadhyay A, Scheffer H, Tilanus MA, Cremers FP, Hoefsloot LH. Clinical and molecular evaluation of probands and family members with familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2009; 50:4379-85. [PMID: 19324841]
  4. Gilmour DF. Familial exudative vitreoretinopathy and related retinopathies. Eye (Lond). 2015; 29:1-14. [PMID: 25323851]
  5. Nikopoulos K, Venselaar H, Collin RW, Riveiro-Alvarez R, Boonstra FN, Hooymans JM, Mukhopadhyay A, Shears D, van Bers M, de Wijs IJ, van Essen AJ, Sijmons RH, Tilanus MA, van Nouhuys CE, Ayuso C, Hoefsloot LH, Cremers FP. Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP. Hum Mutat. 2010; 31:656-66. [PMID: 20340138]
  6. Ye X, Wang Y, Nathans J. The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease. Trends Mol Med. 2010; 16:417-25. [PMID: 20688566]
  7. Ye X, Wang Y, Cahill H, Yu M, Badea TC, Smallwood PM, Peachey NS, Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization. Cell. 2009; 139:285-98. [PMID: 19837032]
  8. Hu H, Xiao X, Li S, Jia X, Guo X, Zhang Q. KIF11 mutations are a common cause of autosomal dominant familial exudative vitreoretinopathy. Br J Ophthalmol. 2016; 100:278-83. [PMID: 26472404]
  9. Collin RW, Nikopoulos K, Dona M, Gilissen C, Hoischen A, Boonstra FN, Poulter JA, Kondo H, Berger W, Toomes C, Tahira T, Mohn LR, Blokland EA, Hetterschijt L, Ali M, Groothuismink JM, Duijkers L, Inglehearn CF, Sollfrank L, Strom TM, Uchio E, van Nouhuys CE, Kremer H, Veltman JA, van Wijk E, Cremers FP. ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature. Proc Natl Acad Sci USA. 2013; 110:9856-61. [PMID: 23716654]
  10. Kashani AH, Learned D, Nudleman E, Drenser KA, Capone A, Trese MT. High prevalence of peripheral retinal vascular anomalies in family members of patients with familial exudative vitreoretinopathy. Ophthalmology. 2014; 121:262-8. [PMID: 24084499]
  11. Seo SH, Yu YS, Park SW, Kim JH, Kim HK, Cho SI, Park H, Lee SJ, Seong MW, Park SS, Kim JY. Molecular Characterization of FZD4, LRP5, and TSPAN12 in Familial Exudative Vitreoretinopathy. Invest Ophthalmol Vis Sci. 2015; 56:5143-51. [PMID: 26244290]
  12. Girirajan S, Campbell CD, Eichler EE. Human copy number variation and complex genetic disease. Annu Rev Genet. 2011; 45:203-26. [PMID: 21854229]
  13. Itsara A, Cooper GM, Baker C, Girirajan S, Li J, Absher D, Krauss RM, Myers RM, Ridker PM, Chasman DI, Mefford H, Ying P, Nickerson DA, Eichler EE. Population analysis of large copy number variants and hotspots of human genetic disease. Am J Hum Genet. 2009; 84:148-61. [PMID: 19166990]
  14. Diskin SJ, Hou C, Glessner JT, Attiyeh EF, Laudenslager M, Bosse K, Cole K, Mossé YP, Wood A, Lynch JE, Pecor K, Diamond M, Winter C, Wang K, Kim C, Geiger EA, McGrady PW, Blakemore AI, London WB, Shaikh TH, Bradfield J, Grant SF, Li H, Devoto M, Rappaport ER, Hakonarson H, Maris JM. Copy number variation at 1q21.1 associated with neuroblastoma. Nature. 2009; 459:987-91. [PMID: 19536264]
  15. Rees E, Kirov G. Copy number variation and neuropsychiatric illness. Curr Opin Genet Dev. 2021; 68:57-63. [PMID: 33752146]
  16. Verbitsky M, Westland R, Perez A, Kiryluk K, Liu Q, Krithivasan P, Mitrotti A, Fasel DA, Batourina E, Sampson MG, Bodria M, Werth M, Kao C, Martino J, Capone VP, Vivante A, Shril S, Kil BH, Marasà M, Zhang JY, Na YJ, Lim TY, Ahram D, Weng PL, Heinzen EL, Carrea A, Piaggio G, Gesualdo L, Manca V, Masnata G, Gigante M, Cusi D, Izzi C, Scolari F, van Wijk JAE, Saraga M, Santoro D, Conti G, Zamboli P, White H, Drozdz D, Zachwieja K, Miklaszewska M, Tkaczyk M, Tomczyk D, Krakowska A, Sikora P, Jarmoliński T, Borszewska-Kornacka MK, Pawluch R, Szczepanska M, Adamczyk P, Mizerska-Wasiak M, Krzemien G, Szmigielska A, Zaniew M, Dobson MG, Darlow JM, Puri P, Barton DE, Furth SL, Warady BA, Gucev Z, Lozanovski VJ, Tasic V, Pisani I, Allegri L, Rodas LM, Campistol JM, Jeanpierre C, Alam S, Casale P, Wong CS, Lin F, Miranda DM, Oliveira EA, Simões ESAC, Barasch JM, Levy B, Wu N, Hildebrandt F, Ghiggeri GM, Latos-Bielenska A, Materna-Kiryluk A, Zhang F, Hakonarson H, Papaioannou VE, Mendelsohn CL, Gharavi AG, Sanna-Cherchi S. The copy number variation landscape of congenital anomalies of the kidney and urinary tract. Nat Genet. 2019; 51:117-27. [PMID: 30578417]
  17. Lauer S, Gresham D. An evolving view of copy number variants. Curr Genet. 2019; 65:1287-95. [PMID: 31076843]
  18. Spielmann M, Lupiáñez DG, Mundlos S. Structural variation in the 3D genome. Nat Rev Genet. 2018; 19:453-67. [PMID: 29692413]
  19. Mayo S, Monfort S, Roselló M, Orellana C, Oltra S, Caro-Llopis A, Martínez F. Chimeric Genes in Deletions and Duplications Associated with Intellectual Disability. Int J Genomics. 2017; 2017:4798474 [PMID: 28630856]
  20. Iwanami M, Oishi A, Ogino K, Seko Y, Nishida-Shimizu T, Yoshimura N, Kato S. Five major sequence variants and copy number variants in the EYS gene account for one-third of Japanese patients with autosomal recessive and simplex retinitis pigmentosa. Mol Vis. 2019; 25:766-79. [PMID: 31814702]
  21. Surl D, Shin S, Lee ST, Choi JR, Lee J, Byeon SH, Han SH, Lim HT, Han J. Copy number variations and multiallelic variants in Korean patients with Leber congenital amaurosis. Mol Vis. 2020; 26:26-35. [PMID: 32165824]
  22. Zampaglione E, Kinde B, Place EM, Navarro-Gomez D, Maher M, Jamshidi F, Nassiri S, Mazzone JA, Finn C, Schlegel D, Comander J, Pierce EA, Bujakowska KM. Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations. Genet Med. 2020; 22:1079-87. [PMID: 32037395]
  23. Whitman MC, Di Gioia SA, Chan WM, Gelber A, Pratt BM, Bell JL, Collins TE, Knowles JA, Armoskus C, Pato M, Pato C, Shaaban S, Staffieri S, MacKinnon S, Maconachie GDE, Elder JE, Traboulsi EI, Gottlob I, Mackey DA, Hunter DG, Engle EC. Recurrent Rare Copy Number Variants Increase Risk for Esotropia. Invest Ophthalmol Vis Sci. 2020; 61:22 [PMID: 32780866]
  24. Huang XY, Zhuang H, Wu JH, Li JK, Hu FY, Zheng Y, Tellier L, Zhang SH, Gao FJ, Zhang JG, Xu GZ. Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy. Mol Vis. 2017; 23:605-13. [PMID: 28867931]
  25. Mammo D, Yonekawa Y, Thomas BJ, Shah AR, Abbey AM, Trese MT, Drenser KA, Capone A. Association of autosomal dominant familial exudative vitreoretinopathy and spinal muscular atrophy. Eur J Ophthalmol. 2015; 25:e116-8. [PMID: 26109022]
  26. Seo SH, Kim MJ, Park SW, Kim JH, Yu YS, Song JY, Cho SI, Ahn JH, Oh YH, Lee JS, Lee S, Seong MW, Park SS, Kim JY. Large Deletions of TSPAN12 Cause Familial Exudative Vitreoretinopathy (FEVR). Invest Ophthalmol Vis Sci. 2016; 57:6902-8. [PMID: 28002565]
  27. 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. 2005; 26:104-12. [PMID: 15981244]
  28. Kashani AH, Brown KT, Chang E, Drenser KA, Capone A, Trese MT. Diversity of retinal vascular anomalies in patients with familial exudative vitreoretinopathy. Ophthalmology. 2014; 121:2220-7. [PMID: 25005911]
  29. Pendergast SD, Trese MT. Familial exudative vitreoretinopathy. Results of surgical management. Ophthalmology. 1998; 105:1015-23. [PMID: 9627651]
  30. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25:1754-60. [PMID: 19451168]
  31. Stenson PD, Mort M, Ball EV, Shaw K, Phillips A, Cooper DN. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet. 2014; 133:1-9. [PMID: 24077912]
  32. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015; 17:405-24. [PMID: 25741868]
  33. Yang L, Kong Y, Dong X, Hu L, Lin Y, Chen X, Ni Q, Lu Y, Wu B, Wang H, Lu QR, Zhou W. Clinical and genetic spectrum of a large cohort of children with epilepsy in China. Genet Med. 2019; 21:564-71. [PMID: 29930392]
  34. Dong L, Wang S, Fu B, Wang J. Evaluation of droplet digital PCR and next generation sequencing for characterizing DNA reference material for KRAS mutation detection. Sci Rep. 2018; 8:9650 [PMID: 30504843]
  35. Robitaille J, MacDonald ML, Kaykas A, Sheldahl LC, Zeisler J, Dubé MP, Zhang LH, Singaraja RR, Guernsey DL, Zheng B, Siebert LF, Hoskin-Mott A, Trese MT, Pimstone SN, Shastry BS, Moon RT, Hayden MR, Goldberg YP, Samuels ME. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet. 2002; 32:326-30. [PMID: 12172548]
  36. Poulter JA, Ali M, Gilmour DF, Rice A, Kondo H, Hayashi K, Mackey DA, Kearns LS, Ruddle JB, Craig JE, Pierce EA, Downey LM, Mohamed MD, Markham AF, Inglehearn CF, Toomes C. Mutations in TSPAN12 Cause Autosomal-Dominant Familial Exudative Vitreoretinopathy. Am J Hum Genet. 2016; 98:592 [PMID: 28863275]
  37. Chen ZY, Battinelli EM, Fielder A, Bundey S, Sims K, Breakefield XO, Craig IW. A mutation in the Norrie disease gene (NDP) associated with X-linked familial exudative vitreoretinopathy. Nat Genet. 1993; 5:180-3. [PMID: 8252044]
  38. Jiao X, Ventruto V, Trese MT, Shastry BS, Hejtmancik JF. Autosomal recessive familial exudative vitreoretinopathy is associated with mutations in LRP5. Am J Hum Genet. 2004; 75:878-84. [PMID: 15346351]
  39. Chen C, Sun L, Li S, Huang L, Zhang T, Wang Z, Yu B, Luo X, Ding X. Novel variants in familial exudative vitreoretinopathy patients with KIF11 mutations and the Genotype-Phenotype correlation. Exp Eye Res. 2020; 199:108165 [PMID: 32730767]
  40. Dixon MW, Stem MS, Schuette JL, Keegan CE, Besirli CG. CTNNB1 mutation associated with familial exudative vitreoretinopathy (FEVR) phenotype. Ophthalmic Genet. 2016; 37:468-70. [PMID: 26967979]
  41. Zhang L, Zhang X, Xu H, Huang L, Zhang S, Liu W, Yang Y, Fei P, Li S, Yang M, Zhao P, Zhu X, Yang Z. Exome sequencing revealed Notch ligand JAG1 as a novel candidate gene for familial exudative vitreoretinopathy. Genet Med. 2020; 22:77-84. [PMID: 31273345]
  42. Coussa RG, Zhao Y, DeBenedictis MJ, Babiuch A, Sears J, Traboulsi EI. Novel mutation in CTNNB1 causes familial exudative vitreoretinopathy (FEVR) and microcephaly: case report and review of the literature. Ophthalmic Genet. 2020; 41:63-8. [PMID: 32039639]
  43. Saitou M, Gokcumen O. An Evolutionary Perspective on the Impact of Genomic Copy Number Variation on Human Health. J Mol Evol. 2020; 88:104-19. [PMID: 31522275]
  44. Jones GE, Ostergaard P, Moore AT, Connell FC, Williams D, Quarrell O, Brady AF, Spier I, Hazan F, Moldovan O, Wieczorek D, Mikat B, Petit F, Coubes C, Saul RA, Brice G, Gordon K, Jeffery S, Mortimer PS, Vasudevan PC, Mansour S. Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR): review of phenotype associated with KIF11 mutations. Eur J Hum Genet. 2014; 22:881-7. [PMID: 24281367]
  45. Kondo H, Matsushita I, Nagata T, Fujihara E, Hosono K, Uchio E, Hotta Y, Kusaka S. Retinal Features of Family Members With Familial Exudative Vitreoretinopathy Caused By Mutations in KIF11 Gene. Transl Vis Sci Technol. 2021; 10:18 [PMID: 34128965]
  46. Shurygina MF, Simonett JM, Parker MA, Mitchell A, Grigorian F, Lifton J, Nagiel A, Shpak AA, Dadali EL, Mishina IA, Weleber RG, Yang P, Pennesi ME. Genotype Phenotype Correlation and Variability in Microcephaly Associated With Chorioretinopathy or Familial Exudative Vitreoretinopathy. Invest Ophthalmol Vis Sci. 2020; 61:2 [PMID: 33137195]
  47. Hartikka H, Mäkitie O, Männikkö M, Doria AS, Daneman A, Cole WG, Ala-Kokko L, Sochett EB. Heterozygous mutations in the LDL receptor-related protein 5 (LRP5) gene are associated with primary osteoporosis in children. J Bone Miner Res. 2005; 20:783-9. [PMID: 15824851]
  48. Boyden LM, Mao J, Belsky J, Mitzner L, Farhi A, Mitnick MA, Wu D, Insogna K, Lifton RP. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med. 2002; 346:1513-21. [PMID: 12015390]
  49. 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. 2012; 18:2438-46. [PMID: 23077402]
  50. Li JK, Li Y, Zhang X, Chen CL, Rao YQ, Fei P, Zhang Q, Zhao P, Li J. Spectrum of Variants in 389 Chinese Probands With Familial Exudative Vitreoretinopathy. Invest Ophthalmol Vis Sci. 2018; 59:5368-81. [PMID: 30452590]
  51. Wu LH, Chen L-H, Xie H, Xie Y-J. Prenatal Diagnosis of a Case of Norrie Disease with Late Development of Bilateral Ocular Malformation. Fetal Pediatr Pathol. 2017; 36:240-5. [PMID: 28394646]
  52. Gruber C, Bogunovic D. Incomplete penetrance in primary immunodeficiency: a skeleton in the closet. Hum Genet. 2020; 139:745-57. [PMID: 32067110]
  53. Piaceri I, Chiari A, Galli C, Bagnoli S, Ferrari C, Saavedra ST, Molinari MA, Vinceti G, Sorbi S, Nacmias B. Incomplete penetrance in familial Alzheimer’s disease with PSEN1 Ala260Gly mutation. Neurol Sci. 2020; 41:2263-6. [PMID: 32328830]
  54. Aref-Eshghi E, Kerkhof J, Pedro VP, Barat-Houari M, Ruiz-Pallares N, Andrau JC, Lacombe D, Van-Gils J, Fergelot P, Dubourg C, Cormier-Daire V, Rondeau S, Lecoquierre F, Saugier-Veber P, Nicolas G, Lesca G, Chatron N, Sanlaville D, Vitobello A, Faivre L, Thauvin-Robinet C, Laumonnier F, Raynaud M, Alders M, Mannens M, Henneman P, Hennekam RC, Velasco G, Francastel C, Ulveling D, Ciolfi A, Pizzi S, Tartaglia M, Heide S, Héron D, Mignot C, Keren B, Whalen S, Afenjar A, Bienvenu T, Campeau PM, Rousseau J, Levy MA, Brick L, Kozenko M, Balci TB, Siu VM, Stuart A, Kadour M, Masters J, Takano K, Kleefstra T, de Leeuw N, Field M, Shaw M, Gecz J, Ainsworth PJ, Lin H, Rodenhiser DI, Friez MJ, Tedder M, Lee JA, DuPont BR, Stevenson RE, Skinner SA, Schwartz CE, Genevieve D, Sadikovic B. Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders. Am J Hum Genet. 2020; 106:356-70. [PMID: 32109418]