with OCA4 (The Human Gene Mutation

Database, HGMD 2013.1). However, this

Glycine 518 residue appears to be con-

served through all available vertebrate

genomes investigated, hence suggesting

a relevant functional role at this position,

plus it is true that other glycine-to-argi-

nine amino acid changes in the SLC45A2

protein have been already associated

with OCA4 (data from HGMD 2013.1:

p.G44R; p.G89R; p.G110R; p.G349R;

p.G370R; p.G404R). Some indirect evi-

dence of an altered association of the

mutant protein with membranes was

also reported by the authors. However, it

is probably expected, for a protein with

12 transmembrane domains, that it

would remain associated with mem-

branes, even in the presence of some

disruptive mutations, although these

minor topological alterations might actu-

ally result in a loss-of-function pheno-

type. The ultimate proof should be

provided by engineering this mutation in

an experimental animal model (i.e.

mouse or zebrafish) and/or, simply, by

reporting its presence in human subjects

diagnosed as OCA4.

Finally, the authors investigated the

landscape where this SLC45A2 muta-

tion was found in Snowflake’s genome

and showed that it is nicely located

within a large run (40 Mbps) of homo-

zygosity, orthologous to human chro-

mosome 5 (where the human

SLC45A2 gene is located), indicating

this allele was inside a block identical

by descent, characteristic for Mende-

lian recessive disorders. Furthermore,

by investigating the patterns of hetero-

zygosity in the Snowflake’s genome,

the authors inferred that it was likely a

result of inbreeding. Through computa-

tional simulations they concluded that

the most probable scenarios for Snow-

flake’s parents were uncle/niece or

aunt/nephew.

It is always rewarding to put an end

to previously unexplained observations,

especially if these refer to remarkable

individuals such this unique albino gor-

illa. Therefore, the authors of this

study must be praised for having com-

pleted the quest for the genetic cause

of the albinism of Snowflake. Person-

ally, as someone born in Barcelona

just 1 yr before Snowflake and some-

one that had the chance to witness

his life through my visits at the Zoo,

as many other visitors worldwide, it is

also good that the conservation plight

of Snowflake’s relatives has been

highlighted; let us hope that this is in

time to contribute to the efforts to

conserve these remarkable but endan-

gered primates.

References

Mart�ınez-Arias, R., Comas, D., Andr�es, A.,

Abell�o, M.T., Domingo-Roura, X., and

Bertranpetit, J. (2000). The tyrosinase

gene in gorillas and the albinism of

‘Snowflake’. Pigment Cell Res. 13, 467–70.

M�artinez-Garc�ıa, M., and Montoliu, L.

(2013). Albinism in Europe. J. Dermatol.

40, 319–24.Newton, J.M., Cohen-Barak, O., Hagiwara,

N., Gardner, J.M., Davisson, M.T., King,

R.A., and Brilliant, M.H. (2001). Muta-

tions in the human orthologue of the

mouse underwhite gene (uw) underlie a

new form of oculocutaneous albinism,

OCA4. Am. J. Hum. Genet. 69, 981–8.Regales, L., Giraldo, P., Garc�ıa-D�ıaz, A.,

Lavado, A., and Montoliu, L. (2003).

Identification and functional validation of

a 5′ upstream regulatory sequence in

the human tyrosinase gene homologous

to the locus control region of the mouse

tyrosinase gene. Pigment Cell Res. 16,

685–92.Roy, R., Cantero, M., and Montoliu, L.

(2004). Molecular and histological analysis

of the albinism of “Snowflake”. In: Pro-

gram and Abstracts of the 12th Meeting

of the European Society for Pigment Cell

Research. Pigment Cell Res. 17, 563–603.

Of white tigers and solute carriers

Alessandro Mongera and Christopher M. Dooleye-mail: alessandro.mongera@tuebingen.mpg.de

The white tiger is a rare variant of the

Bengal tiger (Panthera tigris tigris) rep-

resenting a star attraction in many zoos

around the world. While the earliest

sightings in the jungles of the Indian

subcontinent were recorded in the

1500s, the last report of an individual

seen in the wild dates back to more

than fifty years ago: Indeed, the trade

of exotic animals, trophy hunting, and

habitat destruction have contributed to

heavily reducing the wild population. All

white tigers kept in captivity today are

likely descends of Mohan, a male white

tiger captured in 1951 in the former

State of Rewa, now part of the Repub-

lic of India. Moreover, in an attempt to

maintain the trait, they are highly

inbred, leading to inbreeding depres-

sion-related health problems.

White tigers lack pheomelanin (red-

to-yellow pigment) but have normal

eumelanin (brown-to-black pigment)

production. Vertical black/brown stripes

are formed normally while the orange

background is substituted by a light,

white fur. Furthermore, white tigers

have blue eyes, pink paw pads, and a

pink nose. The genetic basis of this vari-

ant, apart from the monogenetic auto-

somal recessive mode of inheritance,

has been enigmatic so far, although the

similarity with the chinchilla phenotype

in mice have suggested a possible

involvement of classical albino muta-

tions, such as those in the tyrosinase

(TYR) gene.

Xu et al. have recently reported that

the white tiger morph carries a genetic

lesion at a position reported to cause

OCA4 in humans, a form of oculocutane-

ous albinism. First, the authors excluded,

by direct sequencing, the mammalian

coat color modulators MC1R (melano-

cortin 1 receptor), ASIP (agouti-signaling

Coverage on: Xu, X., Dong, G.X., Hu,

X.S., Miao, L., Zhang, X.L., Zhang, D.L.,

Yang, H.D., Zhang, T.Y., Zou, Z.T.,

Zhang, T.T., Zhuang, Y., Bhak, J., Cho,

Y.S., Dai, W.T., Jiang, T.J., Xie, C.,

Li, R., Luo, S.J. (2013). The genetic

basis of white tigers. Curr. Biol. 23(11),

1031–5.

doi: 10.1111/pcmr.12163

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 787

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peptide), TYR (tyrosinase), TYRP1 (tyro-

sine-related protein 1), and SLC7A11

(solute carrier family 7, member 11),

which fail to present any variation associ-

ated with the white tiger phenotype.

Next, using whole-genome sequencing

and restriction-site-associated DNA

sequencing within a known pedigree,

followed by validation in 130 unrelated

tigers by direct sequencing, they identi-

fied an alanine-to-valine substitution in

the solute carrier SLC45A2 as the causa-

tive mutation.

Mutations in SLC45A2, the mouse

underwhite gene (uw), have been linked

to a fourth class of human oculocutane-

ous albinism (OCA4) (Newton et al.,

2001), while the genes defective in the

other classes were already known. For

instance, OCA1 was known to be

associated with mutations in TYR, an

enzyme important for melanin produc-

tion; OCA2, the most common form of

OCA in Africa, with mutations in the

P gene, whose product regulates mela-

nosome pH through ion transport con-

trol; and OCA3, also known as ‘rufous/

red albinism’, with mutations in TYRP1,

encoding another enzyme involved in

pigment biosyn-

thesis.

Identifying the

mutation under-

lying the white

tiger morph rep-

resents an

important

achievement in

that it confirms

how modern

sequencing technology may be fruitfully

used in mapping allelic variants in non-

model organisms and it discloses the

genetic basis of the evocative appear-

ance of an animal that ‘has fascinated

humans for centuries ever since its dis-

covery in the jungles of India’. However,

it has to be acknowledged, as the

authors of this paper do, that genetic

lesions in SLC45A2 have already been

linked to color variation also in medaka

(Fukamachi et al., 2001), horse (Mariat

et al., 2003), chicken, and quail (Gun-

narsson et al., 2007). Recently, also the

Danio rerio albino mutant has been

shown to carry mutations in the coding

sequence of the same solute carrier

(Dooley et al., 2013).

Specifically, medaka fish homozygous

for b (a mutant allele of the gene

SLC45A2) represent a common orange-

red variant that has been bred in Japan

for hundreds of years. In these medaka

as well as in zebrafish albino mutants,

only melanophores are affected, while

the other pigment cell types (yellow

xanthophores, silver iridophores, and

white leukophores) appear normal (Doo-

ley et al., 2013; Fukamachi et al., 2001).

In horses, four ‘base’ colors, that is,

bay, black, brown, and chestnut, con-

tribute to the final coat coloration. Spo-

radically, some of these basic tones can

be diluted, only moderately as in buck-

skin or palomino horses, or strongly as

in cream horses, which have rosy skin,

blue eyes, and a coat that spans from

nearly white to rust-tinged. The ‘cream

mutation’ has been mapped to

SLC45A2 and is part of a set of causal

mutations responsible for coat color var-

iation so far described in horses, includ-

ing genetic lesions in MC1R (leading to

chestnut coat), EDNRB (leading to

white lethal), and ASIP and TYRP (lead-

ing to differential modulation of black

color)(Mariat et al., 2003).

Variation in plumage coloration in

chicken and Japanese quail is affected

by mutations in the Silver locus. Influ-

enced by numerous modifying genes,

these mutations cause a large range of

coat color modifications, but in general

lead to whitish plumage. By a candidate

gene approach, these mutations have

been mapped to the gene SLC45A2.

Interestingly, recessive null alleles

abolish almost completely both forms of

melanin, whereas dominant alleles

carrying missense mutation may

specifically affect only pheomelanin

(Gunnarsson et al., 2007). The pheno-

typic outcomes of these different

mutations, which can be similar in differ-

ent species, may find an explanation

once the exact role of the solute carrier

is fully clarified.

Since its cloning in mouse, SLC45A2

was proposed to function as osmotic

regulator. Sequence similarities with

membrane transporters such as sucrose/

proton symporters in plants and ultra-

structural studies of melanosomes in un-

derwhite mutants indeed suggested an

involvement of this protein in pH control.

Notably, the identity of the substance

transported through the melanosome

membrane remains unclear; cysteine,

necessary for pheomelanin production,

and sugars, cotransported with protons,

are privileged candidates.

Interestingly, Xu et al. argue that the

white tiger variant is a fully viable natural

genetic polymorphism. They reason

that, as mature white tigers have been

spotted or captured in the past, this

character may not affect the overall fit-

ness, excluding the possibility to classify

it as a genetic deformity. It is important

to note that survival to adulthood does

not represent the only parameter to take

in account in the estimation of fitness;

in fact, the defining characteristic of

OCA is the adverse effects of hypopig-

mentation on the visual system which,

although functionally impaired, is still

compatible with adult life (as shown in

numerous species, including humans).

Moreover, it is not clear that natural

genetic polymorphism and disease

(genetic deformity) are necessarily non-

overlapping categories.

Examples of natural color variation

have been recently reported to act

through Kit signaling in stickleback and

agouti in deer mice. It is interesting to

note that the number of reported cases

of natural variation in the key melanin

producing enzyme tyrosinase (Tyr) is

limited, also in humans. Could this be

an indication of an evolutionary con-

straint where less viable variants arise

from mutations in TYR as opposed to

other genes typically identified to be

involved in color variation, for example

SLC45A2, or are the examples still to

be found?

In this regard, zebrafish may offer

some new insights. A recent study on

zebrafish albino has provided evidence

of the involvement of Slc45a2 in melan-

osomal pH homeostasis (Dooley et al.,

2013). First, expression analysis indi-

cates an autonomous role of the pro-

tein within the melanocyte lineage;

second, chemical treatments demon-

strate a role of Slc45a2 in regulating

the normal enzymatic activity of tyrosi-

nase through modulation of proton

exchange, within a molecular axis that

comprises also the organelle acidifier

V-ATPase complex and the potassium-

dependent sodium/calcium exchanger

Slc24a5. Interestingly, ultrastructural

analysis of melanosome biogenesis in

Tyr null mutants reveals a catastrophic

breakdown of melanosomes and toxic-

ity in neighboring cells, whereas various

alleles of slc45a2 simply lead to modu-

lation of melanin content but do not

seem to affect the biochemistry out-

side melanosomes. This could explain

the multiple examples previously

reported in the association of SLC45A2

and color variation among the animal

kingdom.

As we continue to sequence an ever-

greater number of species and variants,

it will be exciting to see what other solu-

tions evolution has come up with to

modify traits and features of our natural

world.

“modernsequencingtechnology maybe fruitfully usedin mappingallelic variants innon-modelorganisms”

788 ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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References

Dooley, C.M., Schwarz, H., Mueller, K.P.,

Mongera, A., Konantz, M., Neuhauss,

S.C., N€usslein-Volhard, C., and Geisler,

R. (2013). Slc45a2 and V-ATPase are

regulators of melanosomal pH homeo-

stasis in zebrafish, providing a mecha-

nism for human pigment evolution and

disease. Pigment Cell Melanoma Res.

26, 205–217.

Fukamachi, S., Shimada, A., and Shima, A.

(2001). Mutations in the gene encoding B,

a novel transporter protein, reduce mela-

nin content in medaka. Nat. Genet. 28,

381–385.Gunnarsson, U., Hellstr€om, A.R., Tixier-

Boichard, M., Minvielle, F., Bed’hom,

B., Ito, S., Jensen, P., Rattink, A., Vereij-

ken, A., and Andersson, L. (2007). Muta-

tions in SLC45A2 cause plumage color

variation in chicken and Japanese quail.

Genetics 175, 867–877.

Mariat, D., Taourit, S., and Guerin, G.

(2003). A mutation in the MATP gene

causes the cream coat colour in the

horse. Genet. Sel. Evol. 35, 119–133.Newton, J.M., Cohen-Barak, O., Hagiwara,

N., Gardner, J.M., Davisson, M.T., King,

R.A., and Brilliant, M.H. (2001). Muta-

tions in the human orthologue of the

mouse underwhite gene (uw) underlie a

new form of oculocutaneous albinism,

OCA4. Am. J. Hum. Genet. 69, 981–988.

Three BRNs are better than two

Colin R. Goding

e-mail: colin.goding@ludwig.ox.ac.uk

Melanoma proliferation is driven by

mutations that activate drivers of the

mitogen-activated kinase (MAPK) path-

way such as NRAS or BRAF. These

mutations occur frequently, but only

rarely go on to progress to full-blown

melanoma. This is because cells faced

with overactive MAPK signalling acti-

vate pro-senescence mechanisms lead-

ing to a largely irreversible cell cycle

arrest as is found in benign nevi.

Only when senescence bypass is

achieved, will cells with MAPK path-

way activating mutations proliferate

indefinitely.

In general, oncogene-induced senes-

cence (OIS) requires both the p53 and

Rb1 pathways to be intact and as such

can be bypassed via many routes.

These include mutation or epigenetic

inactivation of CDKN2A (p16INK4a), over-

expression of cyclin D or b-catenin and

elevated expression of the T-box factors

TBX2 or TBX3. In animal models and in

cell culture, loss of PTEN leading to

constitutive activation of PI3K signalling

also bypasses OIS, while depletion of

the so-called master regulator of the

melanocyte lineage, the microphthalmia-

associated transcription factor (MITF),

promotes senescence.

In the paper from Hohenauer et al., a

new melanoma antisenescence factor is

revealed: BRN3a, a member of the POU

domain transcription factor family that

includes the melanoma-associated tran-

scription factor BRN2 and the pluripo-

tency factor OCT4. BRN3a is required

for the development of neurosecretory

neurons and of endocrine tissues, as

well as the development of subtypes

of sensory neurons. Consistent with its

developmental role, in adults, its

expression appears to be restricted to

subsets of neuronal cells and testis.

However, expression in the melano-

cyte lineage in general and in mela-

noma in particular has not previously

been described.

Hohenauer et al. first show that

BRN3a mRNA is overexpressed in

around 75% of melanoma cell lines

compared with melanocytes, fibroblasts

or keratinocytes, but is not associated

with any particular stage of disease pro-

gression, and examination of tumour

sections revealed a staining pattern

with a variable intensity of either

homogenous or heterogeneous expres-

sion. By using siRNA to deplete BRN3a,

the authors also showed that BRN3a

was important for tumour growth

in vivo in nude mice, with diminished

BRN3a expression leading to a slow-

growth phenotype. The pro-proliferative

effect of BRN3a on melanoma prolifera-

tion in vivo was consistent with results

obtained using cell lines, in which

decreasing BRN3a expression led to

loss of S-phase and, at later times,

increased apoptosis most likely because

BRN3a is a positive regulator of the

anti-apoptotic gene BCL2 (Budhram-

Mahadeo et al., 1999). The cause of

the G1 arrest on BRN3a-depletion

appears to be via the induction of DNA

double-strand breaks, indicated by the

presence of cH2AX foci and consequent

up-regulation of p53 and its target

CDKN1A (p21), a cyclin-dependent

kinase inhibitor. Significantly, lentiviral-

driven ectopic expression of BRN3a in

primary human melanocytes led to

anchorage-independent growth, and

BRN3a could cooperate with BRAFV600E

to transform primary melanocytes, indi-

cating that BRN3a was able to suppress

OIS.

Taken together the data presented

suggest that overexpression of BRN3a

represents a powerful pro-oncogenic

factor. As always, however, the results

lead to several issues that no doubt will

be addressed in the near future. These

include what regulates BRN3a expres-

sion and activity in melanomas, and

when and where it might be expressed

in the melanocyte lineage in develop-

ment and in adults as might be

expected for a factor expressed in

melanoma. But perhaps the most

obvious question is whether BRN3a,

like its close relative BRN2 (Goodall

et al., 2008), represses MITF expres-

sion. Many if not all of the phenotypes

observed would be accounted for if

BRN3a were able to regulate MITF,

because reduced levels of MITF are

known to cooperate with BRAFV600E in

fish melanoma models (Lister et al.,

2013), and changing MITF levels also

leads to alterations in senescence

(Giuliano et al., 2010), and proliferative

Coverage on: Hohenauer et al. (2013).

The neural crest transcription factor

Brn3a is expressed in melanoma and

required for cell cycle progression and

survival. EMBO Mol. Med. 5, 919–934.

doi: 10.1111/pcmr.12150

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 789

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