ARTICLES Analysis of Ocular in Rab38cht/cht Mice

Brian P. Brooks,1,2,3 Denise M. Larson,2,3 Chi-Chao Chan,1 Sten Kjellstrom,4 Richard S. Smith,5,6 Mary A. Crawford,1 Lynn Lamoreux,7 Marjan Huizing,3 Richard Hess,3 Xiaodong Jiao,1 J. Fielding Hejtmancik,1 Arvydas Maminishkis,1 Simon W. M. John,5,6 Ronald Bush,4 and William J. Pavan3

cht PURPOSE. To characterize the ocular phenotype resulting from and RPE thinning. The synergistic effects of the Rab38 and mutation of Rab38, a candidate gene for Hermansky-Pudlak Tyrp1b alleles suggest that TYRP1 is not the only target of syndrome. RAB38 trafficking. This mouse line provides a useful model for cht/cht METHODS. Chocolate mice (cht, Rab38 ) and control het- studying biology and its role in ocular erozygous (Rab38cht/ϩ) and wild-type mice were examined . (Invest Ophthalmol Vis Sci. 2007;48:3905–3913) clinically, histologically, ultrastructurally, and electrophysi- DOI:10.1167/iovs.06-1464 ologically. Mice homozygous for both the Rab38cht and the Tyrp1b alleles were similarly examined. he analysis of mice that exhibit defects in coat coloration cht/cht RESULTS. Rab38 mice showed variable peripheral trans- T(coat color mutants) has aided in the identification of 1 illumination defects at 2 months of age. Patches of RPE hypo- genes important in eye, , and pigmentation. Many of pigmentation were noted clinically in 57% of Rab38cht/cht eyes these genes are mutated in patients with pigmentary anoma- and 6% of Rab38cht/ϩ eyes. Rab38cht/cht mice exhibited thin- lies. Coat color mutants can exhibit a wide range of variation, ning of the iris and RPE and larger b-wave amplitudes in the including altered, dilute, or absent coloration. Skin and hair scotopic range when compared with the control animals. Com- coloration result from the cells in hair follicles (mela- pared with wild-type mice, Rab38cht/cht were nocytes) that synthesize a -based pigment. Melanin is smaller and there were fewer in neuroectodermally derived also produced in the retinal pigment epithelium (RPE) and retinal pigment epithelium; in neural crest-derived choroid of the eye. in skin, hair follicles, and melanocytes, they were smaller in size only. Mutation of both choroid are derived from the neural crest (NC), a transient Rab38 and Tyrp1 produced mice with ocular and coat color population of stem cells that arise early in development at the pigment dilution greater than that seen with either mutation dorsal neural tube. The RPE, in contrast, is derived from the alone. Comprehensive clinical and pathologic analyses showed neuroepithelium. no other organ system or blood defects in Rab38cht/cht mice. Melanin production occurs in specialized organelles within cht/cht pigment cells called melanosomes. The melanin synthetic en- CONCLUSIONS. Rab38 mice show ocular characteristics zymes (including , tyrosinase-related 1 reminiscent of human oculocutaneous , as well as iris (TYRP1), and tautomerase) must be trafficked within the to the melanosome to achieve proper melanin production. Variations in the coat color of mouse From the 1National Eye Institute, the 3National Human Genome Research Institute, and the 4National Institute on Deafness and Other mutants can result from the absence of pigment cells (white Communication Disorders, National Institutes of Health, Bethesda, spots), defects in melanogenic , and aberrant traffick- Maryland; 5Howard Hughes Medical Institute, Bar Harbor, Maine; 6The ing of melanogenic to the melanosome (color alter- Jackson Laboratory, Bar Harbor, Maine; and the 7Comparative Genetics ations or dilution). Program, TexasA&MUniversity, College Station, Texas. Genes mutated in mice with coat color variations have often 2Contributed equally to the work and therefore should be consid- been associated with human pigmentary anomalies with simi- ered equivalent authors. lar phenotypes. These include pigmentary glaucoma,2,3 oculo- Supported by the intramural program of the National Institutes of cutaneous and X-linked ,4–7 Hermansky-Pudlak Health, the Howard Hughes Medical Institute, and National Eye Insti- syndrome (HPS),8 and Chediak-Higashi syndrome. HPS is an tute Grants EY01475 and EY11721. BPB is part of the Joint Physician- Scientist Development Program at the National Eye Institute and the autosomal recessive characterized by variable oculocu- National Human Genome Research Institute. taneous albinism (including foveal hypoplasia, nystagmus, and Submitted for publication December 11, 2006; revised April 30, iris transillumination defects), absent platelet-dense bodies 2007; accepted July 19, 2007. (leading to prolonged bleeding times), and sporadic lung fibro- Disclosure: B.P. Brooks, None; D.M. Larson, None; C.-C. Chan, sis.9,10 Genes for 16 hypopigmented mouse mutants with None; S. Kjellstrom, None; R.S. Smith, None; M.A. Crawford, None; platelet-mediated bleeding defects have been cloned, and mu- L. Lamoreux, None; M. Huizing, None; R. Hess, None; X. Jiao, tations in the human orthologous genes, most of which are None; J.F. Hejtmancik, None; A. Maminishkis, None; S.W.M. John, involved in organelle trafficking, were subsequently found in None; R. Bush, None; W.J. Pavan, None with HPS.11–13 The publication costs of this article were defrayed in part by page b/b charge payment. This article must therefore be marked “advertise- Similarly, Tyrp1 mice carry a mutation in a melanin ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. synthetic and exhibit a coat on a wild-type Corresponding author: Brian P. Brooks, National Eye Institute, strain background. Patients with oculocutaneous albi- 6,14 National Institutes of Health, Building 10, Room 10B16, Bethesda, MD nism type 3 (OCA3) have homozygous TYRP1 mutations, 20892; [email protected]. which result in moderate hypopigmentation. Of course, there

Investigative Ophthalmology & Visual Science, September 2007, Vol. 48, No. 9 Copyright © Association for Research in Vision and Ophthalmology 3905

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can be human and mouse phenotype differences for disruption OH) and an indirect ophthalmoscope (Keeler, Windsor, Berkshire, UK) of the same gene. For example, mutation of Tyrp1 in both with a 90-D condensing lens (Volk, Mentor, OH). The mice were humans and mice results in hypopigmentation and iris transil- euthanatized with carbon dioxide according to institutional guidelines. lumination. However, DBA/2J mice homozygous for the “Young” mice were defined as 2 to 3 months of age; “aged” mice were Tyrp1b allele develop age-dependent iris stromal , ele- defined as older than 1 year. The fundi were also examined. vated intraocular pressure, and pigmentary glaucoma, which For body organ and blood system analysis, three male and three has not been observed in humans with TYRP1 mutations.2,3 female 5-month-old Rab38cht/cht mice were compared with six, age- This difference in known human and mouse phenotypes may and sex-matched wild-type control mice. We analyzed differences in be due to the nature of the mutation and whether or not the organ morphology, serum chemistries, hematocrit, and neutrophil and mutant TYRP1 protein can still stabilize tyrosinase. Because the platelet counts between Rab38cht/cht and wild-type mice. Age-related mouse iris atrophy phenotype is dependent on pigment pro- coat color changes were observed by visual comparison of subgroups duction and known human mutations induce ocular albinism, of mice. it has been suggested that the human mutations are self-rescu- These studies conformed to the principles for laboratory animal ing with respect to iris atrophy.2,3 research outlined by the Animal Welfare Act (National Institutes of The chocolate (Rab38cht/cht) mouse mutant arose sponta- Health/Department of Health and Human Services) and the ARVO neously on the C57BL/6J black background as a dark brown Statement for the Use of Animals in Ophthalmic and Vision Research coat color variant. A G19V point mutation in a highly con- and were approved by the Institutional Animal Care and Use Com- served of Rab38 is responsible for the chocolate mittee. phenotype.15,16 Rab38cht/cht mice have normal blood clotting times. In addition to the chocolate mouse, RAB38 is altered in , Electron Microscopy, and a rat coat color mutant called Ruby (-eyed dilution, R) Melanosome Image Analysis which has been proposed as an animal model for HPS.17 Ruby rats have hypopigmented eyes and coat and a bleeding diathe- For light microscopy, mouse eyes were enucleated and fixed in a phosphate-buffered paraformaldehyde–glutaraldehyde mixture, ac- sis. The Ruby Rab38 translation-initiation codon has a missense 22 mutation that is predicted to stop translation at the first codon cording to published protocols. Hematoxylin and eosin–stained and RAB38 protein is not detected. However, unlike in patients methacrylate-fixed sections from the pupillary–optic nerve axis were with HPS, platelet-dense granules are present with normal used for histopathology. appearance and numbers in Fawn-hooded hypertensive rats, For electron microscopy, mouse eyes were dissected and fixed in which also carry a first codon missense mutation in Rab38 and 4% glutaraldehyde in 0.15% phosphate buffer for 1 hour at room have a Ruby phenotype of hypopigmentation and platelet stor- temperature and then transferred to 4% paraformaldehyde for over- age pool defect.18,19 night fixation at 4°C. Ocular tissue samples were processed through Rabs are small GTP-binding proteins involved in vesicular ascending alcohols, propylene oxide, and a 50:50 mixture of propylene transport, motility, and fusion in the secretory and endocytic oxide and Ladd LX112 epoxy resin. They were then infiltrated with pathways of cells.20,21 The precise function of RAB38 remains 100% LX112 and embedded in fresh resin. Samples were cut in a unknown, although it appears to be important in melanogen- cryotome (Ultracut R; Leica), stained with uranyl acetate and lead esis and necessary for proper targeting of TYRP1 protein in citrate, and examined by (JEM 1010; JEOL, Tokyo, melanosomes.16 Given the role of Rab proteins in trafficking Japan). The density and cross-sectional areas of melanosomes in the RPE and the association of alterations of Rab38 with coat color cht/cht cht/ϩ ϩ/ϩ variants, Rab38cht/cht mice may be a model for an HPS-like and choroid of Rab38 , Rab38 , and Rab38 mice were b/b measured from randomly selected transmission electron microscopy syndrome, or—like Tyrp1 mice—may develop an age-de- ϫ pendent form of pigmentary glaucoma or may display ocular images at 5000 magnification (AxioVision LE, ver. 4.5; Carl Zeiss albinism similar to OCA3. Meditec, Dublin, CA). Care was taken to image comparable areas of the In this study, the ocular phenotype in Rab38cht/cht, posterior pole between different groups of mice. At least three inde- cht/ϩ pendent images were analyzed for each group. RPE areas analyzed per Rab38 , and wild-type mice was assessed by using clinical ␮ 2 examination, histopathology, electrophysiology, and ultra- sample were between 75 and 115 m . structural techniques. Because preliminary evidence suggested that RAB38 is important in TYRP1 targeting, we examined Electrophysiology mice homozygous for both the Rab38cht and Tyrp1b alleles. To Electroretinograms (ERGs) were recorded in 2- to 3-month-old homozy- investigate whether alterations in RAB38 are associated with gous Rab38cht/cht, heterozygous Rab38cht/ϩ, and wild-type (C57BL/6 human disease, RAB38 was sequenced in a small group of Rab38ϩ/ϩ) mice. They were dark-adapted for 12 hours before intra- human subjects with ocular and/or systemic pigmentary abnor- peritoneal anesthesia with ketamine (80 mg/kg) and xylazine (4 mg/ malities. kg). After instillation of 1% proparacaine anesthetic, the pupils were dilated with topical 0.5% tropicamide and 0.5% phenylephrine HCl. Body temperature was maintained near 38°C with a heating pad. ERGs MATERIALS AND METHODS were recorded simultaneously from both eyes, with gold wire loops Mouse Stocks, Clinical Eye, Organ, and placed on the cornea with a drop of methylcellulose. Gold wires were placed on the at the limbus as the differential electrodes, and the Blood Examination ground wire was attached to the left paw. Rab38cht/cht (stock no. 000976) and wild-type C57BL/6J mice (stock Scotopic ERG responses were elicited in the dark-adapted state no. 000664) were obtained from The Jackson Laboratory (Bar Harbor, with single xenon photostrobe flashes (PS33 Photic Stimulator; Grass- ME). Tyrp1b/b mice were supplied by Lynn Lamoreaux (TexasA&M). Telefactor; West Warwick, RI) delivered in a Ganzfeld light-integrating The mice were housed according to our Institutional Animal Review sphere, with interstimulus intervals of 3 to 60 seconds depending on Board standards with a 14-hour light–10-hour dark cycle. Both stimulus intensity. The stimulus intensity range of Ϫ6.9 to ϩ0.6 log Rab38cht/cht and Tyrp1b/b mice are on a C57BL/6 background, mini- cd-s/m2 was obtained with neutral density (ND) filters (Wratten; East- mizing the potential for phenotypic variation due to strain background. man Kodak, Rochester, NY). Responses in a frequency range of 0.1 to Clinical examination of the anterior segment was performed on 1000 Hz (3-dB cutoff) were amplified 5000 times with a 60-Hz line gently restrained, awake mice with a slit lamp (BQ; Haag-Streit, Mason, frequency notch filter (CP511 AC amplifier; Grass-Telefactor). Pho-

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cht/cht FIGURE 1. Rab38 mice, but not Rab38cht/ϩ mice, exhibited pe- ripheral iris transillumination (TI). The degree of TI was graded as ϩ1 (peripheral only, A), ϩ2 (periph- eral and mid-iris, B) or 0 (none, C). Slightly more than 20% of young (2–6 months of age) Rab38cht/cht mice exhibited ϩ2 TI; the remain- der exhibited ϩ1 TI. This propor- tion did not vary in a cohort of older mice (12–24 months), indicat- ing that TI is probably due to vari- able iris hypoplasia, rather than iris atrophy. Rab38cht/ϩ mice showed no iris TI, even at advanced ages (C, age 23 months in this instance). Similarly, wild-type mice did not exhibit iris TI (data not shown).

topic responses were elicited in a light-adapted state on a rod-suppress- 1:100 dilution anti-Rab38 antibody or 1:100 dilution anti-Rab38 and ing white background of 34 cd/m2, with single flashes at 2-second 1:1000 dilution anti-tubulin antibodies (MP Biomedicals, Inc., Aurora, interstimulus intervals. Up to 20 responses were averaged at all inten- IL). A peroxidase-linked secondary antibody was added (GE Health- sities tested. The a-waves were measured from the prestimulus baseline care, Piscataway, NJ) and detected with chemiluminescence (ECL ; to the initial trough. The b-waves were measured either from the GE Healthcare). baseline or from the a-wave trough when present. Implicit times were measured from flash onset to the a- and b-wave maximum. Intensity–response amplitude data were displayed conventionally Human Mutation Analysis on log–log coordinates and log-linear coordinates. Response profiles Research subjects with Hermansky-Pudlak syndrome or with a Her- were compared across intensity range by mixed ANOVA (PROC MIXED in SAS for Windows; ver. 9.0.2; SAS Institute, Inc., Cary, NC). mansky-Pudlak–like syndrome (i.e., patients with oculocutaneous albi- nism and indications of a bleeding defect, but with normal-appearing RAB38 Antibody Production and platelet-dense bodies) and patients with or Protein Detection pigmentary glaucoma consented to venipuncture and research-based DNA analysis. The subjects’ genomic DNA was PCR amplified exon by Polyclonal affinity-purified rabbit anti-mouse Rab38 was prepared exon (primers available on request), with standard techniques,24 and by Bio-Synthesis, Inc. (Lewisville, TX). Rab38 antigen sequence directly sequenced. Automated sequencing was performed (CEQ 2000 LESIEPDIVKPHLTS, position 188-203, was chosen to minimize any sequencer, with CEQ Dye-Terminator Cycle Sequencing kit; Beckman homology with other Rab proteins, using multisequence Rab align- Coulter, Fullerton, CA) according to the manufacturer’s protocols. The ment.23 Western blot analysis was performed on immortalized melanocyte deposited GenBank sequence (NM_022337) was used as a reference cell lines: control melan-Ink4a-1, which produces normal black pig- for identifying polymorphisms (http://www.ncbi.nlm.nih.gov/Gen- ment, and melan-Ink4a-cht5, which is homozygous for Rab38cht/cht bank; provided in the public domain by the National Center for Bio- mutation. Decreasing amounts of both protein preparations were technology Information, Bethesda, MD). loaded on two nondenatured, precast, 4% to 20% Tris- gels All human subject research was conducted with the approval of (Invitrogen, Carlsbad, CA) and transferred to polyvinylidene difluoride local Institutional Review Boards and was in compliance with the (PVDF) membranes (Invitrogen). The membranes were incubated with Declaration of Helsinki.

cht/cht FIGURE 2. Histopathology of Rab38 mice (B, D) revealed iris hypopigmentation and thinning, particularly peripherally (D, arrow) when compared to that in age-matched Rab38cht/ϩ mice (A, C). Wild-type irides were similar to those of Rab38cht/ϩ mice (data not shown). Bars, 10 ␮m.

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cht/cht FIGURE 3. Rab38 mice exhib- ited peripheral patches of depigmen- tation (A, fundus photo). These patches were most frequently found in the superior retina. Transmission electron microscopy showed that they represented focal RPE thinning (B, arrow, border of normal–abnor- mal tissue dissected). Bar, 2 ␮m.

b/b RESULTS be observed in Tyrp1 mice. Specifically, there was no deep- ening of the anterior chamber, ectasia of the cornea, or cup- Hypopigmentation Effects ping of the optic nerve on clinical examination, nor was there Iris: Variable Thinning and Hypopigmentation. We any evidence of significant ganglion cell loss or optic nerve atrophy on histologic sections (data not shown). clinically examined the anterior segments of the eyes of cht/cht cht/cht We performed histopathology on Rab38 and Rab38 mice. Transillumination defects of the irises of cht/ϩ each eye were graded at the slit lamp as 0 (nonexistent), Rab38 mice in both young and old cohorts of mice. The ϩ ϩ irides of young Rab38cht/cht mice showed thinning and hypo- 1 (peripheral; Fig. 1A), and 2 (extending to the central iris; cht/ϩ Fig. 1B). Examination of the anterior segments of young pigmentation compared with age-matched Rab38 control Rab38cht/cht mice (age 1.5–4 months, n ϭ 34 eyes) revealed mice (Fig. 2). Thinning was most pronounced peripherally and ϩ1 and ϩ2 iris transillumination in 79% and 21% of eyes, was variable. Different severities of thinning were often ob- respectively. Transillumination was most prominent peripher- served in the same eye when different sections of the iris were cht/ϩ sampled. Examination of the irides of the aged cohort of ally in all cases. Rab38 mice did not exhibit this pheno- cht/cht type (Fig. 1C, age 1.5–4 months, n ϭ 14 eyes). Rab38 mice showed no qualitative difference from those We aged a cohort of Rab38cht/cht and Rab38cht/ϩ mice until in the young cohort, consistent with our clinical observation they were older than 1 year and performed clinical examina- that there was no atrophic component to this iris thinning tions. Of the aged Rab38cht/cht mouse eyes, 74% and 26% (data not shown). showed ϩ1 and ϩ2 transillumination, respectively (age 12–24 Fundus and RPE: Focal and Thin- months, n ϭ 106 eyes). This result was not significantly differ- ning. Patches of peripheral fundus depigmentation were ent from that observed in the younger cohort of mice (P ϭ noted in 17 (50%) of 34 eyes of the young cohort of 0.83). None of the aged Rab38cht/cht mice developed iris holes Rab38cht/cht mice, usually in the superior quadrant of the ϩ or correctopia, nor was there evidence of pigment dispersion retina (Fig. 3A). In Rab38cht/ mice, 1 (2%) of 42 eyes on the corneal endothelium or the anterior lens capsule. Age- exhibited a similar phenotype, significantly fewer than in matched Rab38cht/ϩ mice continued to show no evidence of Rab38cht/cht mice (P ϭ 8.8 ϫ 10Ϫ8). Similar areas of depig- iris transillumination (n ϭ 28). mentation were present in 30 (57%) of 52 eyes of the aged Although measurement of intraocular pressure was not per- cohort of Rab38cht/cht mice—a rate comparable to that in formed, there was no clinical or histologic evidence of glau- young Rab38cht/cht mice (P ϭ 0.47). Qualitatively, these coma in Rab38cht/cht mice greater than 1 year of age, as would depigmented areas appeared to be of similar size and char-

FIGURE 4. Melanosomes were smaller in cross-sectional area and fewer in the RPE of Rab38cht/cht mice (A) com- pared with wild-type (Rab38ϩ/ϩ; C); heterozygote (Rab38cht/ϩ) mice were similar to wild-type (B). Melanosomes in Rab38cht/cht mice were able to enter the apical villi of RPE cells (A, arrow- heads), suggesting that, unlike RAB27a, RAB38 is not necessary for transport into these structures. Mela- nosomes from the NC-derived cho- roid were also smaller in Rab38cht/cht mice than in wild-type mice. Bar, 2 ␮m.

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melanosomes, the Rab38cht/ϩ mice showed no significant dif- ference in the size or number of choroidal melanosomes when compared with wild-type (Figs. 4E, 4F; Table 1). Levels of RAB38 Protein. To determine the amount of RAB38 protein in wild-type and Rab38cht/cht cells, we devel- oped an anti-RAB38 antibody. On Western blot, anti-RAB38 antibody recognizes a single, correctly sized protein band in a mouse melanocyte cell line (Fig. 6). The mouse skin melano- cyte cell lines we used are homozygous for the Ink4a null mutation, to allow for rapid immortalizing, and are from the same inbred mouse background (C57BL/6). Therefore, melan- Ink4a-1 cells and melan-Ink4a-cht5 cells should differ only at the Rab38 locus. Melan-Ink4a-cht5 cells homozygous for the Rab38cht allele showed much-reduced RAB38 protein expres- sion on Western blot analysis. Electrophysiology Consistent with Albinism. Electro- retinography was performed on 2- to 3-month-old wild-type (C57BL/6), Rab38cht/ϩ, and Rab38cht/cht animals. All three animal groups had normal a- and b-wave morphology and timing (data not shown). Compared with wild-type animals, Rab38cht/cht animals showed a statistically significant increase in b-wave amplitude under moderate-intensity, scotopic condi- tions (Fig. 7A). There was no statistically significant difference in a-wave amplitude (scotopic or photopic, data not shown) or FIGURE 5. The distribution of melanosome cross-sectional area in the photopic b-wave (Fig. 7B). RPE and choroid of Rab38cht/cht mice demonstrates the significantly Coat Color Change with Age. Coat color pigment dilution smaller areas of Rab38cht/cht melanosomes relative to wild-type. was obvious in the light-brown coats of very young (ϳ1 month old) Rab38cht/cht mice, unlike the dark coats of Rab38cht/ϩand wild-type littermates. At approximately 3 months of age, the cht/cht acter in the young and old Rab38 mice. Wild-type Rab38cht/cht coat color had darkened, but remained distin- C57BL/6J mice do not exhibit these areas of hypopigmenta- guishable from the black coats of the Rab38cht/ϩ and wild-type tion (n ϭ 50). Transmission electron micrographs of the littermates (Fig. 8). junction between normal-appearing and depigmented RPE in Rab38cht/cht mice showed focal hypoplasia of the RPE Tyrp1 and Rab38 (Fig. 3B). cht/cht Altered Melanosomes in Choroid and RPE. We exam- Rab38 mice were mated to mice homozygous for the ined RPE and choroid melanosomes in 3-month-old brown (b) allele of Tyrp1, and the obligate heterozygous off- cht/cht cht/ϩ spring were then intercrossed to create double-homozygous Rab38 , Rab38 , and wild-type C57BL/6J eyes by elec- cht/cht b/b cht/cht cht/cht Rab38 ; Tyrp1 mice. Because both the Rab38 tron microscopy. The RPE of Rab38 mice showed fewer b/b and smaller mature (stage IV) melanosomes than did the RPE of mice and the Tyrp1 mice had a C57BL/6 background, any Rab38cht/ϩand wild-type mice (Figs. 4A–C, 5; Table 1). Those change in pigmentation was presumably due to the effect of cht/cht the mutant alleles. Double homozygous mice (age, 2–3 mature melanosomes that are present in Rab38 mice, ϭ however, appeared capable of entering the apical villi of the months) had striking iris transillumination (n 8 eyes) com- cells. Immature melanosomes did not appear to accumulate in any of the samples examined. The melanosomes of the NC–derived melanocytes in the choroid in the Rab38cht/cht mice were smaller in cross-sec- tional area than were those of the wild-type mice (Figs. 4D, 4F, 5; Table 1). The number of melanosomes, however, was un- changed by the presence of the Rab38cht allele. As with RPE

TABLE 1. Comparison of Melanosomes in RPE and Choroid

Melanosome Density Melanosome Area Genotype (n/␮m2) (␮m2)

RPE Rab38cht/cht 0.48 Ϯ 0.12 (5)* 0.06 Ϯ 0.04 (120)* Rab38cht/ϩ 1.08 Ϯ 0.29 (4) 0.13 Ϯ 0.11 (220) Rab38ϩ/ϩ 0.98 Ϯ 0.08 (4) 0.12 Ϯ 0.08 (255) Choroid Rab38cht/cht 2.79 Ϯ 0.72 (4) 0.04 Ϯ 0.03 (748)* Rab38cht/ϩ 3.83 Ϯ 1.48 (3) 0.07 Ϯ 0.04 (481) Rab38ϩ/ϩ 2.63 Ϯ 0.75 (4) 0.07 Ϯ 0.04 (646) FIGURE 6. An immortalized pigmented mouse melanocyte cell line Data are the mean Ϯ SD (sample number). melan-Melan-Ink4a-cht5 had a reduced amount of RAB38 protein com- * Group mean differs from wild-type mean at P Ͻ 0.01, by one-way pared with the normal black pigmented cell line, melan-Ink4a-1, on ANOVA with the Dunnett posttest for significance. Western blot.

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cht/cht cht/ϩ FIGURE 7. Scotopic (A) and photopic (B) ERG b-wave amplitudes (ϮSEM from 2.5-month-old homozygous (Rab38 ), heterozygous (Rab38 ), and wild-type C57BL/6J (Rab38ϩ/ϩ) mice. Compared with wild-type mice, homozygous mice had significantly larger b-wave amplitudes in the scotopic range (ANOVA, P Ͻ 0.01). In the photopic range, there was no significant difference between any of the groups. Homozygous and wild-type: n ϭ 5 animals/10 eyes; heterozygous: n ϭ 2 animals/4 eyes.

pared with age-matched Rab38cht/cht and Tyrp1b/b mice, as phism, c.T105C, was observed in subjects with pigmentary well as a lighter coat color (Fig. 9). Histologic examination glaucoma, HPS and an HPS-like syndrome. This polymorphism showed significantly reduced pigmentation, especially in the causes no change in amino acid sequence (p.S35S) and is not neuroectodermally derived pigmented tissue (e.g., the poste- predicted to alter splicing. In one individual with pigmentary rior pigmented layer of the iris and the RPE). glaucoma, a heterozygous c.C583A change was identified that is predicted to cause a p.P195T change. This is well- cht/cht Rab38 and Human Disease conserved across mammalian species (mouse, rat, chimpanzee, Blood and Organ Systems. We clinically and pathologi- cow, and dog). cally analyzed three male and three female 5-month-old cht/cht Rab38 mice and compared them with six age- and sex- DISCUSSION matched wild-type control mice (Table 2). Organ morphology, serum chemistries, hematocrit, and platelet counts were not Study of mouse coat color mutants has provided many insights different between Rab38cht/cht and wild-type mice. The neu- into human pigmentation function and disorders. The ocular trophil count in Rab38cht/cht mice was significantly higher than hypopigmentation defect of the pigmentation dilution that in wild-type mice, but within the normal range for other Rab38cht/cht mouse was characterized and analyzed, to provide mouse strains (e.g., FVB). additional information about Rab38’s possible role in human Sequencing of RAB38 in Patient Samples. All three ex- diseases such as ocular albinism, Hermansky-Pudlak syndrome, ons and the intron–exon boundaries of RAB38 were se- or pigmentary glaucoma. quenced in 12 subjects with unclassified, nonsyndromic OCA We examined the anterior segments, RPE, and other aspects phenotypes, 9 subjects with HPS, 12 with HPS-like (oculocu- of the eyes of Rab38cht/cht mice. The results showed that taneous albinism [OCA] with bleeding abnormalities, but nor- Rab38cht/cht mice display iris and RPE thinning and focal de- mal platelet dense bodies), and 17 with pigmentary glaucoma. pigmentation as well as ocular phenotypes similar to those of Subjects with OCA did not have coding-region mutations in the human OCA. For example, electroretinography performed on known genes for these disorders. A single-nucleotide polymor- young Rab38cht/cht mice is reminiscent of findings in some

cht/cht FIGURE 8. In one-month-old Rab38 mice, the light brown coat color was easily distinguished from the black coat of wild-type and Rab38cht/ϩ littermates. In 3-month-old and older Rab38cht/cht mice, the darker coat was still distin- guishable from the black coat of the wild-type and Rab38cht/ϩ littermates.

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Krill and Lee postulate that this supranormal ERG is the result of increased internal reflection of light within the eye from reduced pigmentation. This response, however, has not been observed in all patients with albinism.26–28 The ocular pigment dilution in Rab38cht/cht mice is most likely due to the presence of fewer and smaller melanosomes in the RPE and/or smaller melanosomes in the choroid. Our analysis suggests that the role of Rab38 is more pronounced in neuroectodermally derived structures, such as the RPE, than in NC-derived tissues, such as the choroidal melanocytes. Al- though the precise reason for this difference is unclear, recent experiments by Wasmeier et al.29 have shown that Rab32 and Rab38 may have redundant roles in melanogenesis. A tissue- specific difference in the effect of the Rab38 mutation could therefore be related to a difference in Rab32 expression. Although we did not quantitate melanosome size and number in older animals, we suspect that the age-related increase in pigmentation observed in these mice is related to compensa- tory changes in melanocytes with time. Similar increasing pig- mentation has been noted clinically in some patients with OCA.30 In the Tyrp1b/b mouse, the number of RPE and choroidal melanosomes is decreased,31 and the Tyrp1b/b melanosomes are smaller and rounder than in the wild-type mouse.32 Other data suggest the Rab38 is involved in appropriate targeting of TYRP1 within melanocytes,16 and the Rab38cht/cht mouse brown coat and on a black strain background resem- bled the Tyrp1b/b phenotype. Murine Tyrp1 functions enzy- matically in the synthesis of eumelanin (black melanin), and melanin formed in Tyrp1b/b mutant is brown.33–36 Additional functions of Tyrp1 include stabilization of the melanin-synthe- sizing enzyme, tyrosinase, in melanosomes, and maintenance of melanosomal structure. To investigate the relationship of Rab38 and Tyrp1 in vivo, we created mice homozygous for the Rab38cht allele and the Tyrp1b allele. The double-mutant mice showed a striking loss of pigmentation that was qualitatively greater than the sum of hypopigmentation observed in Rab38cht/cht Tyrp1ϩ/ϩ and Rab38ϩ/ϩTyrp1b/b mice. This finding suggests that there is not a simple linear relationship between RAB38 function and TYRP1 function. If the sole role of RAB38 was to target TYRP1 to melanosomes properly, we would expect that disruption of RAB38 function would have little effect on the Tyrp1b/b phe-

TABLE 2. Results of Systemic Analysis b/b FIGURE 9. The Tyrp1 allele interacted synergistically with the cht/cht Rab38 allele in C57BL/6 mice, to produce striking pigment dilu- Test Rab38cht/cht Rab38؉/؉ P tion. Slit lamp examination of the anterior segments of (A)Rab38ϩ/ϩ, ϩ/ϩ cht/cht ϩ/ϩ ϩ/ϩ b/b Tyrp1 ;(B) Rab38 , Tyrp1 ;(C) Rab38 , Tyrp1 ; and RBC count 8.4 10 0.15 cht/cht b/b (D) Rab38 , Tyrp1 mice showed significantly more depigmen- Hemoglobin 12.9 13.9 0.18 tation and iris transillumination in the double-mutant homozygote than Hematocrit 40.2 42.7 0.17 in the other three strains. This effect was observed in both the eyes and Platelets 1016 837 0.18 cht/cht b/b the coat color of the mice (G). (E, F) Rab38 , Tyrp1 mice Neutrophils 0.259 0.075 0.002 showed reduced pigmentation on histologic sections, particularly in Cholesterol 117 85 0.058 the neuroectodermally derived layers. This difference in pigmentation BUN 21 33 0.03 was evident in the posterior pigmented epithelial layer of the iris (E, Magnesium 1.17 1.45 0.1 ␮ arrowhead) and the RPE (F, arrowhead). Bars, 50 m. Alkaline phosphatase 63.6 102 0.1 Total bilirubin 0.18 0.32 0.07 humans with albinism. Krill and Lee25 describe supranormal GGT 1.66 4.16 0.15 ERG responses under scotopic conditions in patients with oculocutaneous or ocular albinism. Photopic and flicker fusion Test data are the mean of six mice in each group. Histopathology cht/cht responses were normal, however, and carriers of X-linked for Rab38 : two of three females had suppurative metritis, albinism were not significantly different from those in control thought to be postpartum; two of six mice had suppurative otitis media; two of six had mild lymphocytic infiltrates in nasal mucosa; one subjects. They also observe that the scotopic changes in the of six had mild lymphocytic laryngitis; and one of six had chronic albino ERG tend to normalize with age, perhaps due to in- pancreatitis. Histopathology for Rab38ϩ/ϩ: four of six had mild gran- creased pigmentation over time. Because our measurements ulocytic cholecystitis; one of six had uterine endometrial edema; one were conducted in the mice at 3 months of age, they are most of six had edema of tympanic membrane; and one of six had focal likely to model the findings in younger patients with albinism. lacrimal gland adenitis.

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notype, as both proteins would function in the same “linear” Sequencing of the RAB38 gene in patients with OCA who pathway to produce pigment. Our observations that the double did not have mutations in the known albinism genes did not mutants were significantly hypopigmented implies that TYRP1 reveal any sequence changes. Suzuki et al.38 likewise did not is not the only protein involved in RAB38-mediated pigment, find any RAB38 mutations in their cohort of Japanese patients particularly in the neuroectodermally derived layers of the eye. with albinism. We therefore conclude that RAB38 is not a The double mutants may have such a dramatic phenotype major locus for human OCA. The fact remains, however, that because of misrouting of proteins other than TYRP1 to the the Rab38cht allele affects coat color; as such, it is still quite melanosomes. possible that RAB38—although not itself a major disease lo- Tyrp1b/b mice on a DBA/2J background develop iris stromal cus—modifies the phenotype in patients with pigment-related atrophy and pigmentary glaucoma with age.2,3 Iris transillumi- diseases. nation, which is observed in Rab38cht/cht mice, can be seen in iris atrophy as well as in hypopigmentation. To address this, we examined the irides of an aged cohort of Rab38cht/cht mice and Acknowledgments found no qualitative difference from a young cohort. There was no evidence in young or aged Rab38cht/cht of significant The authors thank Richard King’s laboratory, University of Minnesota, iris atrophy, pigment dispersion, or glaucoma. Therefore, the for clinical data; Dot Bennett for the melan-Ink4a-1 and melan-Ink4a- iris transillumination in Rab38cht/cht mice was more likely due cht5 cells used in the study; the National Human Genome Research to hypopigmentation and/or hypoplasia of the iris rather than Institute (NHGRI) mouse core for rederivation of mouse lines; and John to iris atrophy. The possibility remains that a Rab38 mutation Hammer, Xufeng Wu, and members of the Pavan laboratory for useful could cause a pigmentary glaucoma phenotype on other discussions. mouse genetic backgrounds and/or could act as a modifier allele of this disease. References In one patient with pigmentary glaucoma, the Rab38 se- quence showed a heterozygous c.C583A change, which is 1. Bennett DC, Lamoreux ML. The color loci of mice: a genetic predicted to cause a p.P195T change. Whether this change is century. Pigment Cell Res. 2003;16:333–344. disease-related is unclear. This proline is well-conserved across 2. Chang B, Smith RS, Hawes NL, et al. Interacting loci cause severe mammalian species (mouse, rat, chimpanzee, cow, and dog). iris atrophy and glaucoma in DBA/2J mice. Nat Genet. 1999;21: Although a proline-to- change is likely to be signifi- 405–409. cant, the fact that Rab38cht/ϩ mice appeared identical with 3. Anderson MG, Smith RS, Hawes NL, et al. Mutations in genes wild-type mice and that Rab38cht/cht mice did not develop encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice. Nat Genet. 2002;30:81–85. pigmentary glaucoma argues against heterozygous changes 4. Brilliant MH. The mouse p (-eyed dilution) and human P having primary pathologic consequences in humans. genes, oculocutaneous albinism type 2 (OCA2), and melanosomal Two lines of evidence suggest that Rab38 mutation may pH. Pigment Cell Res. 2001;14:86–93. cause human Hermansky-Pudlak syndrome. First, the rat HPS 5. Newton JM, Cohen-Barak O, Hagiwara N, et al. Mutations in the model Ruby (red-eyed dilution, R) hypopigmentation and human orthologue of the mouse underwhite gene (uw) underlie a bleeding phenotype is caused by an Rab38 mutation. No new form of oculocutaneous albinism, OCA4. Am J Hum Genet. RAB38 protein is produced in the Ruby rat.17 In addition, 2001;69:981–988. Rab38, like other Rabs, is probably involved in vesicular traf- 6. Sarangarajan R, Boissy RE. Tyrp1 and oculocutaneous albinism ficking, and several known HPS genes are intracellular vesicle type 3. Pigment Cell Res. 2001;14:437–444. trafficking proteins.9,16 7. Samaraweera P, Shen B, Newton JM, Barsh GS, Orlow SJ. The We did not observe any blood or organ system defects in mouse ocular albinism 1 gene product is an endolysosomal pro- the Rab38cht/cht mice. Neutrophil counts were significantly tein. Exp Eye Res. 2001;72:319–329. higher than in wild-type mice, but within the normal range for 8. Li W, Rusiniak ME, Chintala S, et al. 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