Study of mouse coat color mutants has provided many insights into human pigmentation function and disorders. The ocular hypopigmentation defect of the pigmentation dilution Rab38cht/cht mouse was characterized and analyzed, to provide additional information about Rab38’s possible role in human diseases such as ocular albinism, Hermansky-Pudlak syndrome, or pigmentary glaucoma.
We examined the anterior segments, RPE, and other aspects of the eyes of Rab38cht/cht
mice. The results showed that Rab38cht/cht
mice display iris and RPE thinning and focal depigmentation as well as ocular phenotypes similar to those of human OCA. For example, electroretinography performed on young Rab38cht/cht
mice is reminiscent of findings in some humans with albinism. Krill and Lee25
describe supranormal ERG responses under scotopic conditions in patients with oculocutaneous or ocular albinism. Photopic and flicker fusion responses were normal, however, and carriers of X-linked albinism were not significantly different from those in control subjects. They also observe that the scotopic changes in the albino ERG tend to normalize with age, perhaps due to increased pigmentation over time. Because our measurements were conducted in the mice at 3 months of age, they are most likely to model the findings in younger patients with albinism. 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. Although the precise reason for this difference is unclear, recent experiments by Wasmeier et al.29
have shown that Rab32
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 compensatory changes in melanocytes with time. Similar increasing pigmentation 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 eye color on a black strain background resembled the Tyrp1b/b
phenotype. Murine Tyrp1
functions enzymatically 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-synthesizing 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 Tyrp1+/+ and Rab38+/+Tyrp1b/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 phenotype, as both proteins would function in the same “linear” pathway to produce pigment. Our observations that the double mutants were significantly hypopigmented implies that TYRP1 is not the only protein involved in RAB38-mediated pigment, particularly in the neuroectodermally derived layers of the eye. The double mutants may have such a dramatic phenotype because of misrouting of proteins other than TYRP1 to the melanosomes.
mice on a DBA/2J background develop iris stromal atrophy and pigmentary glaucoma with age.2,3
Iris transillumination, 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 found no qualitative difference from a young cohort. There was no evidence in young or aged Rab38cht/cht
of significant iris atrophy, pigment dispersion, or glaucoma. Therefore, the iris transillumination in Rab38cht/cht
mice was more likely due to hypopigmentation and/or hypoplasia of the iris rather than to iris atrophy. The possibility remains that a Rab38
mutation could cause a pigmentary glaucoma phenotype on other mouse genetic backgrounds and/or could act as a modifier allele of this disease.
In one patient with pigmentary glaucoma, the Rab38 sequence showed a heterozygous c.C583A change, which is predicted to cause a p.P195T change. Whether this change is disease-related is unclear. This proline is well-conserved across mammalian species (mouse, rat, chimpanzee, cow, and dog). Although a proline-to-threonine change is likely to be significant, the fact that Rab38cht/+ mice appeared identical with wild-type mice and that Rab38cht/cht mice did not develop pigmentary glaucoma argues against heterozygous changes having primary pathologic consequences in humans.
Two lines of evidence suggest that Rab38
mutation may cause human Hermansky-Pudlak syndrome. First, the rat HPS model Ruby (red-eyed dilution, R
) hypopigmentation and bleeding phenotype is caused by an Rab38
mutation. No RAB38 protein is produced in the Ruby rat.17
In addition, Rab38
, like other Rabs, is probably involved in vesicular trafficking, and several known HPS genes are intracellular vesicle trafficking proteins.9,16
We did not observe any blood or organ system defects in the Rab38cht/cht
mice. Neutrophil counts were significantly higher than in wild-type mice, but within the normal range for other mouse strains (e.g., FVB). Further analysis is necessary to determine whether this difference results in any impairment of the immune system. Unlike the Ruby rat, RAB38 is produced in Rab38cht/cht
mice, which would be predicted for a point-mutation protein, but the steady state level of mutant RAB38 is much reduced. Recent in vitro work suggests that the chocolate allele produces functionally inactive RAB38.29
These results, along with data showing that Rab38cht/cht
mice have normal bleeding times16,37
suggest that the Rab38cht/cht
allele does not lead to the systemic abnormalities seen in HPS.
Our preliminary analysis of the RAB38 gene in a small number of patients with HPS or an HPS-like condition revealed only a single nucleotide change that does not result in amino acid substitution or clearly alter a splice site. In addition, this same nucleotide change was observed in patients with pigmentary glaucoma. A detailed systemic evaluation of the Rab38cht/cht mouse failed to reveal any abnormality beyond ocular and cutaneous pigmentation dilution. Specifically, the Rab38cht/cht mouse did not have the platelet dysfunction that is characteristic of HPS. Future studies of the functional significance of the Rab38cht allele and the human sequence change could be performed to elucidate the potential role of RAB38 in HPS. Alternatively, RAB38 could be involved in another form of syndromic albinism not covered in our patient sample.
Sequencing of the RAB38
gene in patients with OCA who did not have mutations in the known albinism genes did not reveal any sequence changes. Suzuki et al.38
likewise did not find any RAB38
mutations in their cohort of Japanese patients with albinism. We therefore conclude that RAB38
is not a major locus for human OCA. The fact remains, however, that the Rab38cht
allele affects coat color; as such, it is still quite possible that RAB38
—although not itself a major disease locus—modifies the phenotype in patients with pigment-related diseases.