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The cell adhesion gene PVRL3 is associated with congenital ocular defects

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Abstract

We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell–cell adhesion mechanism in human ocular development.

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Acknowledgments

We thank the subject and his relatives for participating in the Developmental Genome Anatomy Project and Heather Ferguson, Chantal Kelly and Shahrin Ahsan for assistance as study coordinators. This work was supported by NIH grants 5R01EY10123-15 (RLM), 5R01HD060050-02 (RLM), 5P01GM061354-07 (CCM, RLM, JFG, BJQ), R01EY021505 (SAL), R01EY12995 (MLR), and T35-EY07151 (QTD). All experiments comply with the current laws of the countries (US, Israel and Japan) in which they were performed.

Conflict of interest

The authors declare no conflicts of interest.

Author information

Author notes

  1. Anne W. Higgins

    Present address: Departments of Hospital Laboratories and Pathology, UMass Memorial Medical Center and UMass Medical School, Worcester, MA, 01605, USA

  2. Irfan Saadi

    Present address: Department of Anatomy and Cell Biology, KU Medical Center, Kansas City, KS, 66160, USA

Authors and Affiliations

  1. Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    Salil A. Lachke, Irfan Saadi, Qiongchao Xi & Richard L. Maas

  2. Department of Biological Sciences, University of Delaware, Newark, DE, 19716, USA

    Salil A. Lachke

  3. Departments of Obstetrics, Gynecology and Reproductive Biology and of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    Anne W. Higgins, Michelle Long, Bradley J. Quade & Cynthia C. Morton

  4. Radiation Research Center for Frontier Science Research, Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan

    Maiko Inagaki

  5. Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, 02114, USA

    Michael E. Talkowski & James F. Gusella

  6. Department of Genetics, Harvard Medical School, Boston, MA, 02114, USA

    Michael E. Talkowski & James F. Gusella

  7. Department of Pediatrics, Keck School of Medicine, University of Southern California Medical Center, Los Angeles, CA, 90033, USA

    Atsuko Fujimoto

  8. Department of Zoology, Miami University, Oxford, OH, 45056, USA

    Michael L. Robinson

  9. Center for Human and Molecular Genetics, Columbus Children’s Research Institute, Columbus, OH, 43205, USA

    Ying Yang

  10. The Ohio State University College of Optometry, Columbus, OH, 43210, USA

    Quynh T. Duong

  11. The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, 52900, Ramat-Gan, Israel

    Irit Shapira & Benny Motro

  12. Department of Molecular Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, 537-8511, Japan

    Jun Miyoshi

  13. Division of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan

    Yoshimi Takai

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  1. Salil A. Lachke
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Corresponding author

Correspondence to Richard L. Maas.

Additional information

S. A. Lachke and A. W. Higgins contributed equally to this work.

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Supplemental data include five figures and one table and can be found with this article online.

Supplemental Fig. 1. Nek7 hypomorphic mutant mice do not exhibit lens defects. (A) Reverse Transcriptase (RT)-PCR analysis of E12.5 mouse embryonic ocular tissue from wild type (+/+), heterozygous (±), and homozygous (-/-) littermates for the Sanger Nek7 gene trap allele demonstrates that the gene trap generates a hypomorphic Nek7 allele. (B) Quantitative RT-PCR analysis of E12.5 mouse embryonic ocular tissue from wild type (+/+), heterozygous (±), and homozygous (-/-) littermates for the Sanger Nek7 gene trap allele confirms that the gene trap generates a hypomorphic Nek7 allele. (C-E) Staining for β-galactosidase activity in (C) wild type (WT), (D) heterozygous (Nek7 ±), and (E) homozygous (Nek7-/-) for the Sanger Nek7 gene trap allele that contains a knock-in LacZ transgene allele. (F–H) Staining for β-galactosidase activity in mouse Nek7-/- embryos homozygous for the Sanger Nek7 gene trap allele at (F) E10.5, (G) E11.5, and (H) E12.5 demonstrates increased LacZ transgene expression in the eye as development occurs. Lens abnormality or cataract was not detected in Nek7-/- hypomorphic mice at embryonic (above) and adult stages (data not shown), or in Nek7 nullizygotes.

Supplemental Fig. 2. Nek7 null mutant mice do not exhibit lens defects at post-natal data 5 (P5). (A) H&E stained section of P5 wild type eye. (B) H&E stained section of P5 Nek7-/- mutant eye demonstrates the absence of an obvious lens or ocular defect. Abbr.: Co, cornea; Le, lens; Rt, retina.

Supplemental Fig. 3. Nectin 3 is expressed in the developing lens and prospective ciliary epithelia. (A) Immunofluorescence microscopy (IFM) of E14.5 mouse eye demonstrates Nectin 3 expression (red) in the AEL. (A’) High magnification of boxed region in (A). Polarized staining of Nectin 3 in the AEL is indicated by arrow. (B) IFM of E13.5 mouse embryos demonstrates Nectin 3 expression in the presumptive ciliary epithelium. PE, Pigment epithelium. (B’) High magnification of boxed region in (B). Nectin-3 staining indicated by arrow. (C) IFM of E14.5 mouse embryos demonstrates Nectin 3 expression in the presumptive ciliary epithelium. NPE, Non-pigmented epithelium. (C’) High magnification of boxed region in (C). Polarized staining of Nectin 3 in NPE is indicated by arrow. (D) IFM of E18.5 mouse embryos at E18.5 demonstrates expression of Nectin 3 in the presumptive ciliary epithelium. (D’) High magnification of boxed region in (D). Polarized Nectin 3 expression in NPE is indicated by arrow. Other abbr.: AEL, Anterior epithelium of the lens; FC, Fiber cells. Scale bars: (A) 15 μm, (A’) 8 μm, (B) 16 μm, (B’) 6 μm, (C) 10 μm, (C’) 7 μm, (D) 15 μm, (D’) 10 μm.

Supplemental Fig. 4. The ari mutation maps to the Pvrl3 locus and affects Pvrl3 expression in the prospective ciliary epithelia. (A) Genetic intervals between ari and the closest polymorphic markers were determined by examining the phenotype in recombinant backcross animals out of a total of 656 backcross progeny. Mice used to draw the conclusion that ari is located between D16Mit126 and D16Mit61 are summarized in Supplementary Table 1. (B) Physical distance encompassing the ari genetic interval according to mouse genome databases. The physical interval where ari lies is approximately 2.3 Mb. The Pvrl3 gene is approximately 109 kb. The distance between D16Mit126 and the 5’ end of Pvrl3 is 415 kb, and the distance between D16Mit61 and the 3’ end of Pvrl3 is 1.8 Mb. (C) Hematoxylin-stained section demonstrates morphology and (D) section in situ analysis on ocular region of wild type littermate mouse demonstrates normal Pvrl3 expression (arrow) in E15.5 presumptive ciliary epithelia. (C’) Hematoxylin-stained section demonstrates morphology and (D’) section in situ analysis on ocular region of an E15.5 ari mouse mutant demonstrates background levels of Pvrl3 expression (asterisk) in the presumptive ciliary epithelium. The retina region is outlined by dashed line. Abbr.: npe, non pigment epithelium; pe, pigment epithelium; r, retina.

Supplemental Fig. 5. Chromosome 3 breakpoint separates five putative long-distance regulatory elements upstream of PVRL3. (A) Schematic representation of human chromosome 3 with red box showing region highlighted in (B). (B) Chromatin Immunoprecipitation followed by DNA sequencing (ChIP-seq) analysis in eight human cell lines identifies several loci (green boxes) that display Histone 3 lysine 4 mono-methylation (H3K4Me1) and histone 3 lysine 4 acetylation (H3K27Ac) of the chromosome 3 genomic region upstream of PVRL3 and the chromosomal 3 breakpoint (dashed red line) which is separated from the PVRL3 transcription unit by patient DGAP113’s translocation. However, no significant signal for tri-methylated Histone 3 lysine 4 (H3KMe3) in this genomic region is observed, also compatible with the presence of long-range regulatory elements. In constrast, significant tri-methylated Histone 3 lysine 4 (H3KMe3) signals (blue boxes) are observed only near the PVRL3 and DPPA4 transcription start sites, indicative of promoter-driven regulatory elements.

Supplemental Table 1 Summary of the genetic backcross analysis performed to map the ari mutation. Abbr.: C: Cataract; H: Heterozygous for the microsatellite polymorphism (FVB/N and C57BL/6); N: Normal eye; F: Homozygous for the FVB/N polymorphism. Microsatellite polymorphisms are arranged left to right in a centromeric to telomeric orientation on mouse chromosome 16. Note: Mice used to draw the conclusion that ari is located between D16Mit126 and D16Mit61 are summarized in red letters. Mouse (“Animal”) 598 demonstrates that the mutation must be telomeric to D16Mit126. Mouse 694 demonstrates that the mutation must be telomeric to D16Mit59. Mouse 143 demonstrates that the mutation must be telomeric to D16Mit84. Mouse 272 demonstrates that the mutation must be centromeric to D16Mit61. Mouse 611 demonstrates that the mutation must be telomeric to D16Mit84. Mouse 695 demonstrates that the mutation must be telomeric to D16Mit126.

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Lachke, S.A., Higgins, A.W., Inagaki, M. et al. The cell adhesion gene PVRL3 is associated with congenital ocular defects. Hum Genet 131, 235–250 (2012). https://doi.org/10.1007/s00439-011-1064-z

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