Ophthalmic Surgery, Lasers and Imaging Retina

Case Report 

Insights into Retinal Development Using Live Imaging in Female Carriers of Choroideremia

Katherine J. Wert, PhD; Benjamin Bakall, MD, PhD; Alexander G. Bassuk, MD, PhD; Stephen H. Tsang, MD, PhD; Vinit B. Mahajan, MD, PhD

Abstract

Lineage tracing can provide key insights into the development of tissues, such as the retina. Yet it is not possible to manipulate human cells during embryogenesis. The authors observed a distinct phenotype in female carriers of X-linked disorders, in particular, carriers of choroideremia caused by mutations in CHM, encoding Rab escort protein-1. The authors found that X chromosome inactivation provides a method for retinal lineage tracing in human patients. Live imaging of female carriers displays a developmental pattern that is different within the peripheral retina compared with the posterior retina and provides important insights into the development and migration of retinal cells.

[Ophthalmic Surg Lasers Imaging Retina. 2019;50:e158–e162.]

Abstract

Lineage tracing can provide key insights into the development of tissues, such as the retina. Yet it is not possible to manipulate human cells during embryogenesis. The authors observed a distinct phenotype in female carriers of X-linked disorders, in particular, carriers of choroideremia caused by mutations in CHM, encoding Rab escort protein-1. The authors found that X chromosome inactivation provides a method for retinal lineage tracing in human patients. Live imaging of female carriers displays a developmental pattern that is different within the peripheral retina compared with the posterior retina and provides important insights into the development and migration of retinal cells.

[Ophthalmic Surg Lasers Imaging Retina. 2019;50:e158–e162.]

Introduction

Lineage-tracing tools have allowed for the understanding of cell fate determination and progenitor cell contribution during embryonic and postnatal development.1–6 Ideally, mapping the fate of clonal populations of cells that carry genes with mutations leading to developmental and degenerative disorders could provide important information on the mechanism of the disease alleles. However, mammalian species used in research may not accurately reflect the developmental patterns of tissues and cell fate determination that occurs in humans.

We found that X-linked disorders have a distinct phenotype in asymptomatic female carriers that could provide key insights into retinal development. In particular, we examined patients from multiple unrelated families with genetic mutations in the CHM gene, encoding the Rab escort protein(REP)-1 protein. These mutations cause X-linked choroideremia, a progressive retinopathy beginning in childhood, with significant visual loss by middle age.7,8 Here we show how the pattern of degeneration may reflect developmental properties of the retina.

Case Report

Family A

A 26-year-old woman was evaluated for fundus pigmentary changes. She was asymptomatic with 20/20 vision in both eyes and no family history of retinal dystrophy (Figure 1A, II-I). On examination, distinct linear streaks of intraretinal pigment were observed throughout the peripheral retina, anterior to the equator (Figure 2A, arrows). Rounded clumps of intraretinal pigment were noted in the posterior retina (Figure 2B, arrowhead). Interestingly, these pigmented clumps were surrounded by regions of non-pigmentation (Figure 2B, asterisks). Examination of her asymptomatic 50-year-old mother and 22-year-old sister showed similar pigmentary lines in the anterior peripheral retina (Figures 2C and 2D, arrows). Examination of the 18-year-old brother (Figure 1A, II–III) contributed to a diagnosis of choroideremia with characteristic fundus features and clinical presentation. Molecular analysis revealed a pathogenic variant in the CHM gene causing an p.Arg293Stop change in the REP-1 protein. The mutation was present in a hemizygous state in the affected male and was heterozygous for all females.

Pedigrees for choroideremia. (A) In the first family, the mother (I-2) and two daughters (II-1 and II-2) are carriers of the mutation in the CHM gene located on the X chromosome and display pigmented streaks upon fundus examination. The son (II-3) is hemizygous for the CHM mutation and has clinical manifestation of the choroideremia phenotype. The only granddaughter is unaffected (III-1). (B) A second family has a mother (I-2) that is a carrier of the mutation in CHM with pigmented streaks and has passed on the carrier status and the pigmentation phenotype to her daughters (II-3 and II-5) and the choroideremia phenotype to each of her three sons (II-1, II-2, and II-4). (C–E) Three additional families also have a mother (I-2) that is a carrier for the CHM mutation and displays the pigmentation carrier phenotype. Each of the sons display the clinical choroideremia phenotype and disease pathology (II-1). Squares = males; circles = females; open symbols = unaffected; filled symbols = affected; circles with black dot and bar = female carriers with pigmented streaks in the retina

Figure 1.

Pedigrees for choroideremia. (A) In the first family, the mother (I-2) and two daughters (II-1 and II-2) are carriers of the mutation in the CHM gene located on the X chromosome and display pigmented streaks upon fundus examination. The son (II-3) is hemizygous for the CHM mutation and has clinical manifestation of the choroideremia phenotype. The only granddaughter is unaffected (III-1). (B) A second family has a mother (I-2) that is a carrier of the mutation in CHM with pigmented streaks and has passed on the carrier status and the pigmentation phenotype to her daughters (II-3 and II-5) and the choroideremia phenotype to each of her three sons (II-1, II-2, and II-4). (C–E) Three additional families also have a mother (I-2) that is a carrier for the CHM mutation and displays the pigmentation carrier phenotype. Each of the sons display the clinical choroideremia phenotype and disease pathology (II-1). Squares = males; circles = females; open symbols = unaffected; filled symbols = affected; circles with black dot and bar = female carriers with pigmented streaks in the retina

Clinical phenotype of female carriers for choroideremia. (A) Linear streaks of intraretinal pigment in the peripheral retina anterior to the equator in an asymptomatic 26-year-old woman heterozygous for a pathogenic variant in the CHM gene, causing an Arg293Stop in the Rab escort protein-1 protein. (B) This same individual had pigment clumping in the mid-peripheral retina, with regions lacking pigment accumulation. (C) Linear streaks of pigmentation were visible in the peripheral retina of both her 50-year-old mother and (D) 22-year-old sister, both asymptomatic carriers of the CHM mutation. (E) A similar pattern of linear pigmentation streaks anterior to the equator were detectable in a 42-year-old woman from another family that is heterozygous for a 4-base pair deletion of AGTC at position -2 of intron 4, affecting a splice site in the CHM gene. Both pigment clumping in the mid-peripheral retina and regions lacking pigment accumulation were also visible in this individual. (F) Retinal pigment clumping and (G) pigmented linear streaks were detected in her 7-year-old and 11-year-old daughters, heterozygous for the mutation disrupting CHM, displaying the early onset of this carrier phenotype. (H) Schematic drawing representing the findings of pigment accumulation in female carriers of X-linked choroideremia. Clonal cell lineages can be followed using in vivo imaging in the eye to detect cells with the CHM mutation (pigmented circles) and cells that are wild type for CHM (open circles) based on X-chromosome inactivation skewing within the retina, early in development. Arrows, pigmented linear streaks in the peripheral retina; arrowheads, pigment clumping in the mid-peripheral retina; asterisks, sites of wild-type CHM retinal cells lacking pigmentation.

Figure 2.

Clinical phenotype of female carriers for choroideremia. (A) Linear streaks of intraretinal pigment in the peripheral retina anterior to the equator in an asymptomatic 26-year-old woman heterozygous for a pathogenic variant in the CHM gene, causing an Arg293Stop in the Rab escort protein-1 protein. (B) This same individual had pigment clumping in the mid-peripheral retina, with regions lacking pigment accumulation. (C) Linear streaks of pigmentation were visible in the peripheral retina of both her 50-year-old mother and (D) 22-year-old sister, both asymptomatic carriers of the CHM mutation. (E) A similar pattern of linear pigmentation streaks anterior to the equator were detectable in a 42-year-old woman from another family that is heterozygous for a 4-base pair deletion of AGTC at position -2 of intron 4, affecting a splice site in the CHM gene. Both pigment clumping in the mid-peripheral retina and regions lacking pigment accumulation were also visible in this individual. (F) Retinal pigment clumping and (G) pigmented linear streaks were detected in her 7-year-old and 11-year-old daughters, heterozygous for the mutation disrupting CHM, displaying the early onset of this carrier phenotype. (H) Schematic drawing representing the findings of pigment accumulation in female carriers of X-linked choroideremia. Clonal cell lineages can be followed using in vivo imaging in the eye to detect cells with the CHM mutation (pigmented circles) and cells that are wild type for CHM (open circles) based on X-chromosome inactivation skewing within the retina, early in development. Arrows, pigmented linear streaks in the peripheral retina; arrowheads, pigment clumping in the mid-peripheral retina; asterisks, sites of wild-type CHM retinal cells lacking pigmentation.

Family B

We observed a similar pattern of peripheral retinal pigment streaks anterior to the equator in a 42-year-old woman (Figure 2E, arrows). Pigment clumping (arrowheads) and streaks without pigmentation (asterisks) were visible with funduscopy. Her 7-year-old and 11-year-old daughters displayed retinal pigment clumping in the posterior retina (Figure 2F, arrowheads) and linear streaks in the anterior peripheral retina (Figure 2G, arrows). Her three sons exhibited typical findings of choroideremia (Figure 1B). We identified a 4-base pair deletion of AGTC at position -2 of intron 4, involving a splice site of the CHM gene. This mutation was present in a hemizygous state in affected males and was heterozygous for all females.

Families C Through E

In three other families, similar peripheral retinal pigment streaks were seen in both eyes of the mothers of sons with choroideremia (Figures 1C to 1E).

Discussion

In summary, we describe female carriers with molecularly confirmed X-linked choroideremia who exhibit streaks of hyperpigmentation in the peripheral anterior retina. Striated patchy pigmentary changes in the retinal periphery were previously observed in female carriers of X-linked ocular albinism.10 It is possible that the carrier pigmentation phenotype seen in X-linked ocular albinism could be non-pigmented retinal pigment epithelial cells that have migrated into the peripheral fundus; however, X-linked retinitis pigmentosa carriers have also shown a pigmentation phenotype upon examination.11,12 Thus, the linear streaks are presumed to be formed as a result of random inactivation of one of the X chromosomes in subsets of retinal progenitor cells in the early fetal development, according to the Lyon hypothesis.10 X chromosome inactivation (XCI) is a mechanism used in mammals to inactivate either the maternal or paternal X chromosome to equilibrate the expression of X-linked genes across genders.13–15 In choroideremia patients, these lines are also presumed to occur as part of XCI and are used as a tool to facilitate the diagnosis of choroideremia in affected families.8,11,16–23

XCI in retinal degenerative disorders has been studied using mammalian research models. In mice with a lacZ transgene inserted on the X chromosome, it was found that cells from both populations migrated during development and formed clonal patches of XCI in a radial pattern.13 After birth, columns of lacZ active and inactive cells were visible in the retina. In a canine model of X-linked retinitis pigmentosa, carrier females showed patches of rod opsin mislocalization within the posterior retina.13 However, the methods of XCI during retinal development in mammalian research models may not reflect the human XCI and retinal cell fate determination. For instance, there are clear differences in XCI between human and mouse cells. In mouse cells, the paternally inherited X chromosome becomes silenced at the four- to eight-cell stage, then is reactivated in epiblast cells for XCI to occur during implantation.24 The silencing of the paternal X chromosome does not occur in human embryos. Furthermore, the X chromosomes in mouse epiblasts do not express Xist, which is an effector of XCI, but both X chromosomes in human preimplantation embryos have been shown to express Xist.24

In humans, our observations suggest a developmental pattern that is different in the peripheral retina compared with the posterior retina. We find that non-pigmented (open circles) and pigmented streaks (colored circles) in the peripheral retina suggest a possible radial migration pattern of cells and their progeny, whereas within the posterior pole cells and their progeny remain clustered together with no long-distance migration patterns, as seen by the clusters of pigmented cells (closed circles) within the mid-retinal region (Figure 2H). We have observed a method that acts as an in vivo retinal lineage tracing in human patients, without surgical or experimental manipulations. Analysis of the healthy, non-pigmented cells in comparison to the pigmented cells carrying the CHM mutation in the asymptomatic female carriers can provide insight into the cells and their progeny that lead to choroideremia disease progression. As these retinal progenitor cells divide and migrate throughout the retina, the pigmentary degeneration labels a specific cell lineage and reveals how cells develop in the retina. Additionally, the difference in the patterning from healthy and diseased cells in the peripheral retina (formation of streaks) compared with the posterior retina (clusters of pigmentation) using live human imaging of these marked retinal cells during childhood to adulthood in choroideremia-affected families could provide important insight into the development and migration of retinal cells during eye development.

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Authors

From Byers Eye Institute, Omics Laboratory, Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, California (KJW, VBM); Associated Retina Consultants, Phoenix, Arizona (BB); University of Arizona College of Medicine, Phoenix, Arizona (BB); the Department of Pediatrics, University of Iowa, Iowa City, Iowa (AGB); Bernard and Shirlee Brown Glaucoma Laboratory, Department of Pathology and Cell Biology, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York (SHT); and Palo Alto Veterans Administration, Palo Alto, California (VBM).

Drs. Mahajan and Bassuk are supported by NIH grants (R01EY026682, R01EY024665, R01EY025225, R01EY024698, R21AG050437, P30EY026877) and Research to Prevent Blindness (RPB), New York, New York. The Barbara & Donald Jonas Laboratory of Regenerative Medicine and Bernard & Shirlee Brown Glaucoma Laboratory are supported by the National Institute of Health (5P30EY019007, R01EY018213, R01EY024698, R21AG050437), National Cancer Institute Core (5P30CA013696), the RPB Physician-Scientist Award, and unrestricted funds from RPB. Dr. Tsang is a member of the RD-CURE Consortium and is supported by the Tistou and Charlotte Kerstan Foundation, the Schneeweiss Stem Cell Fund, New York State (C029572), the Joel Hoffman Fund, the Professor Gertrude Rothschild Stem Cell Foundation, and the Gebroe Family Foundation. The remaining authors report no relevant financial disclosures.

Address correspondence to Vinit B. Mahajan, MD, PhD, Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University School of Medicine, Palo Alto, CA 94304; email: vinit.mahajan@stanford.edu.

Received: February 24, 2018
Accepted: June 04, 2018

10.3928/23258160-20190503-15

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