The Roxi DBE^CEL gene and the Seymour DBE^ALT gene are the best studied at present. They have been bred and studied over multiple generations and breeders have tested their cats' hearing as well as observing any physical abnormalities. Most physical anomalies are cosmetic, but some have reported deformed or absent tear ducts. Whether this is associated with one gene or with mixed genes isn't yet known. Where it did occur, it was strongly linked to the blue eye as odd-eyed DBE cats were only affected on the blue side. More than 50 heterozygous French Celestial Shorthairs (Altai gene) had their hearing tested and none were found to be deaf. Only the homozygous Celestials showed bilateral deafness so blue-eye to blue-eye matings were prohibited in this breed.

In contrast, a German breeder of DBE Maine Coons (undisclosed mutation) has found that her DBE Maine Coons with full colour can be deaf and this is not uncommon. Other breeders have had deaf full-colour Maine Coons from the same line. The DBE Maine Coon from this line has the wider-spaced eyes or telecanthus when compared to one with normal eye colour. when I look at the photos of the two cats in blue, I have to say that the blue-eyed one looks different. The eye spacing is characteristic of the mutation which means it is unlikely to improve with each generation of breeding. Although the mutation used was not disclosed, this deafness in full-colour DBE cats has been seen in the Aktau blue-eyed mutation (tracing to a cat called Caspian Black Hole). Some Aktau lines were bred to Barnaul mutation lines (founded by Velvet Slavicat). There is also the Rociri Elvis mutation which seems to be a mutation (in the Netherlands) of the "normal" white spotting gene. Some breeders have used the Rociri Elvis gene (a "latent" DBE gene that originated in his mother's line) as the basis and sometimes mixing with Altai or another mutation to improve eye colour!

A PAX3 INSERTION IN THE CELESTIAL BREED AND CERTAIN FELINE BREEDING LINES WITH DOMINANT BLUE EYES
Marie Abitbol, Alice Couronne, Caroline Dufaure de Citres, Vincent Gache
Animal Genetics. 2024;00:1–6., 9 April 2024

The authors found that a mutation of Paired Box 3 (PAX3) on chromosome C1 causes the minimal white spotting (sometimes no visible white spotting) and dominant blue eyes (DBE) in the French Celestial breed. PAX3 is a key regulator of MITF (Melanocyte-Inducing Transcription Factor) and PAX3 mutations cause white spotting with or without blue eyes in other species. An endogenous retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 was found.

All 52 Celestial and Celestial-mixed cats with the DBE phenotype had the mutation. Their 22 non-DBE littermates and 87 non-DBE cats from various breeds (control samples) did not have the mutation. The Celestial founder, Roxi, was heterozygous for the mutation. It was also found in 9 DBE Maine Coon cats related to Roxi and four DBE Siberian cats with an uncertain origin. The mutation shows a dominant inheritance pattern and is not to associated with deafness. It has been termed DBE^CEL (Celestial Dominant Blue Eyes) and originated from Roxi. Another Topaz founder, Seymour, does not have the DBE^CEL mutation; Seymour's mutation has yet to be determined.

Kittens with minimal white spotting and no blue eye but with a red-eye effect during infancy are known as latents (LBE). Latent cats possess the DBE^CEL mutation without expressing the blue eye phenotype. The French registry LOOF place restrictions on the Celestial breeding program: all British Shorthair/Longhairs used in breeding must test negative for white gloving, white spotting, and dominant white alleles (all are KIT gene mutations). Colourpoint and silver gene (inhibitor) cats are also not allowed to breed. Because of existing concerns linked to blue eyes and white spotting/masking, DBE Celestials have been tested for deafness. Out of 47 DBE Celestial cats tested, there were 3 cases of deafness and these were on one side only. These cats had medical histories of ear problems unrelated to DBE.

PAX3 mutations in humans are associated with Waardenburg syndrome characterised by a white forelock, premature greying, deafness and heterochromia or brilliant blue eyes. No auditory or premature greying is been found in the Celestial breed. [Sarah's note: these were found when 2 different genes were bred together in the Topaz. The Seymour gene is no longer in Celestial cats]

DBE in the Celestial breed is an autosomal dominant trait with variable expression and incomplete penetrance for both white spotting and blue eyes, and pleiotropy (the gene affects other processes in the organism, and tissue development in the embryo). A single Celestial DBE x DBE mating was performed, resulting in three healthy kittens and a presumed homozygous white kitten that died from cleft palate just after birth.

PAX3 HAPLOINSUFFICIENCY IN MAINE COON CATS WITH DOMINANT BLUE EYES AND HEARING LOSS RESEMBLING THE HUMAN WAARDENBURG SYNDROME [**ROCIRI ELVIS LINEAGE]
Gabriela Rudd Garces, Daniela Farke, Martin J Schmidt, Anna Letko, Katja Schirl, Marie Abitbol, Tosso Leeb, Leslie A Lyons, Gesine Luhken, G3 Genes|Genomes|Genetics, 2024;, jkae131, PAX3 haploinsufficiency in Maine Coon cats with dominant blue eyes and hearing loss resembling the human Waardenburg syndrome Published: 13 June 2024

Dominant blue eyes (DBE) with minimal white spotting cats, are characterized by pigmentary abnormalities of the iris and skin, and in some cases, sensorineural hearing loss. This is due to abnormal distribution of melanocytes during embryo development, leading to a lack of melanocytes in the eyes and skin. The causative genetic variant has been found in the PAX3 gene, which affects (among other things) the differentiation of melanoblasts into melanocytes. DBE cats are characterised by striking blue eyes (one or both) in combination with variable white patches of fur. "Latent DBE cats" have minimal white spotting and no blue eyes but display a red-eye effect during infancy.

The genetic variant known as DBE^RE ("Rociri Elvis" gene) causes DBE with deafness. The phenotype of the heterozygotes resembles human Waardenburg syndrome type 1 and the Splashed white in horses. It seems likely that homozygous DBE^RE is lethal in the womb. Special examination methods (BAER test) is needed to determine if there is impaired hearing. DNA testing to identify the DBE^RE variant can easily clarify whether a DBE phenotype cat has a gene variant that is associated with deafness.

The DBE^RE test identified the substitution of 1 base pair c.937C>T in the PAX3 gene.
n/n = homozygous normal = Wild type: does not have the PAX3: c.937C>T nonsense variant (DBE^RE); if the tested cat has the DBE phenotype it is caused by a different genetic variant.
n/ DBE^RE = heterozygous for the mutation = DBE with potential deafness. One copy of DBE^RE variant, 50% of offspring will inherit the variant and potential deafness.
DBE^RE / DBE^RE = homozygous for the variant gene, not viable (die in utero or at birth).

A Maine Coon breeder had reported multiple deaths in several litters from 2020 to 2023 and observed signs of deafness in blue-eyed cats. These all traced to the Rociri Elvis line (Dutch line). All blue-eye cats showed hearing abnormalities, but normal green-eyed cats did not. 58 cats from two different DBE lineages (48 from the Dutch Rociri Elvis line and 10 from the Topaz lines), were sampled. None of the cats had the dominant white KIT gene mutation, but some had white spotting related to the KIT gene.

The 48 cats from the Rociri Elvis bloodline included 9 green-eyed and 31 blue-eyed cats, some of the later having impaired hearing. There were 8 stillborn kittens, but cause of death was not related to presence of the PAX3 mutation. A single copy of the mutant allele (PAX3: c.937C>T) was found to cause DBE, impaired hearing loss and minimal white spotting. Samples from the Topaz lineage included 10 blue-eyed animals; 8 cats were BAER tested and showed signs of impaired hearing.

All 31 blue-eyed cats from the Dutch lineage were heterozygous for the mutant PAX3:c.937C>T nonsense variant, while all 9 green-eyed cats were homozygous wildtype. The Topaz lines did not have this mutation.

The German Animal Protection Law prohibits the breeding of animals with defective hearing. The Rociri Elvis (DBE^RE) and Topaz (Seymour line) genes have led to multiple DBE breeding programmes. The researches advocated implementation of PAX3 variant testing for all DBE cats in order to select suitable mating partners, avoid mixing DBE mutations and avoid the mutations strongly linked to impaired hearing.

DIFFERENT FOUNDING EFFECTS UNDERLIE DOMINANT BLUE EYES (DBE) IN THE DOMESTIC CAT [**SEYMOUR DESCENDANTS]
Marie Abitbol, Caroline Dufaure de Citres, Gabriela Rudd Garces, Gesine Luehken, Leslie A. Lyons and Vincent Gache. Animals 2024, 14, 1845 (21 June 2024)

Simplified summary
Whole-genome sequencing of a DBE cat revealed an RD-114 retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 (namely NC_018730.3:g.206975776_206975777insN[433]) known to contain regulatory sequences. This variant was fully associated with DBE in two British lineages, in Altai cats, and in some other DBE lineages. The authors propose that this NC_018730.3:g.206975776_206975777insN[433] variant represents the DBE-ALT (Altai Dominant Blue Eye) allele in the domestic cat.

Unlike the extinct American Ojos Azules, homozygous mutant kittens were viable with no deformities. They had large white spots or white fur, and some had impaired hearing. Altai breeders found that certain cats have the DBE variant but do not express the blue eye phenotype ("latents").
NC_018730.3:g.206975776_206975777insN[433] - also called RD-114 LTR, or RD, in literature.
NC_018730.3:g.206974029_206974030insN[395] - also called FERV1 LTR, or FERV1, in the literature.
Pleiotropy means a gene that has multiple effects.

Altai (founder: Fyodor) - Autosomal dominant, incomplete penetrance, variable expressivity, homozygotes viable with Van-like pattern or solid white. NC_018730.3:g.206975776_206975777insN[433] variant found.
Topaz (Founders: Roxi and Seymour) - Autosomal dominant, incomplete penetrance, variable expressivity and pleiotropy, presumed homozygotes/compound heterozygotes viable with Van-like pattern or solid white, some with deafness. (Because these are founders of several lines, I suspect they had both NC_018730.3:g.206974029_206974030insN[395] (Roxi) and NC_018730.3:g.206975776_206975777insN[433] (Seymour) variants which interacted to create the "black-eyed" cats, "panda pattern" cats and progressive greying, one variant was later eliminated from the breeding programme.)
Celestial (Founder: Roxi) - Autosomal dominant, incomplete penetrance, variable expressivity and pleiotropy; homozygotes solid white, not viable (only one born, died at birth). NC_018730.3:g.206974029_206974030insN[395] found.
British DBE (Seymour line) - Autosomal dominant, incomplete penetrance, variable expressivity and pleiotropy; no homozygous kittens have been produced. NC_018730.3:g.206975776_206975777insN[433] variant found.
British DBE (Nanotigr line, founders: Oliver and his daughter Amelia) - Autosomal dominant, incomplete penetrance, variable expressivity; homozygotes have Van-like pattern or solid white and some are deaf. NC_018730.3:g.206975776_206975777insN[433] variant found.
British DBE (Igor line, founder: Igor Azur Dream) - Autosomal dominant, some deaf cats reported, incomplete penetrance, variable expressivity, and pleiotropy, no known homozygotes.
British DBE (Nadeya line, spontaneous variant, founder: Nadeya Ermine Trace) - Autosomal dominant, some deaf cats reported, incomplete penetrance, variable expressivity, and pleiotropy, no known homozygotes.
Persian DBE (Alaska line, founder: Seymour) - Autosomal dominant, incomplete penetrance, variable expressivity, no homozygotes found for study. NC_018730.3:g.206975776_206975777insN[433] variant found.
Persian DBE (Cyrridwen line, founder: Marusya) - Autosomal dominant, incomplete penetrance, variable expressivity, no homozygotes found for study.
Ragdoll DBE (Founder: Seymour) - Autosomal dominant, incomplete penetrance, variable expressivity, no homozygotes found for study. NC_018730.3:g.206975776_206975777insN[433] variant found.
Sphynx DBE (Founder: Unknown) - Autosomal dominant, incomplete penetrance, variable expressivity, no homozygotes found for study. NC_018730.3:g.206975776_206975777insN[433] variant found.
Siberian DBE (Founder: Unknown) - Autosomal dominant, incomplete penetrance, variable expressivity, no homozygotes found for study. NC_018730.3:g.206974029_206974030insN[395] found.
Maine Coon DBE (Topaz line, founders: Roxi and Seymour) - Autosomal dominant, incomplete penetrance, variable expressivity, no known homozygotes. NC_018730.3:g.206974029_206974030insN[395] found.
Maine Coon DBE (spontaneous variant, Dutch line, founder: Rociri Elvis) - Autosomal dominant, incomplete penetrance, variable expressivity and pleiotropy; no known homozygotes. PAX3:c.937C>T nonsense variant
Maine Coon (Pillowtalk line, founder unknown) - Autosomal dominant, no homozygotes found for study.
Maine Coon (Nahal line, founder Nahal (domestic cat from Russia)) - no data available.

A shared RD-114 LTR insertion among the Altai and the Seymour and Nanotigr British lineages suggests a common ancestor for these three breeding lines.
The DBE mutation inherited from Seymour is called DBE-ALT. He is considered (by testing) the Altai cat, which means that Roxi - whom breeders considered an Altai - is now considered (through testing) not to be an Altai. Igor, previously assumed to be an Altai cat, tested negative for DBE-ALT and is not genetically Altai.
Homozygous DBE-ALT/DBE-ALT are viable and show no abnormalities except deafness for some of them. A single homozygous DBE-CEL/DBE-CEL kitten and a single putative compound heterozygous DBE-CEL/DBE-RE kitten have been reported; both showed limb abnormalities and contractures, abnormal head shape, and died at birth. (My own communications about the Topaz breed during its development, not included in this paper a no cats were available for testing [the cattery was dispersed due to the war in Ukraine], indicate compound heterozygous DBE-ALT/DBE-CEL cats were viable, but had unusual distribution of white, dark-blue or black eyes, premature greying and deafness because the head is wholly white.)

CONSIDERATIONS FOR ALL BLUE-EYED BREEDS

There are various known and unknown genes involved in white spotting and in blue-eyes. In solid white and high white cats there is a link to deafness because melanin is involved in the development of the inner ear (the part that detects sound vibrations). BAER testing (acoustically evoked brain stem responses) is used by many European breeders to ensure that they don't deliberately breed deaf cats. The prevalence of deafness and partial hearing in an experimental colony of white cats was 67% (deaf - 0.55 coefficient of heritability) and 29% (partial hearing - 0.75 coefficient of heritability) which suggests a pleiotropic major gene (a gene that has 2 seemingly unrelated traits) and the likelihood of polygenes.

Geigy CA, Heid S, Steffen F, Danielson K, Jaggy A, Gaillard C (2007). "Does a pleiotropic gene explain deafness and blue irises in white cats?". Veterinary Journal. 173 (3): 548 553. PMID 16956778.

This was commented upon by Strain GM, in "Deafness in blue-eyed white cats: the uphill road to solving polygenic disorders."Vet J. 2007 May;173(3):471-2. Epub 2007 Feb 21.

Until you are able to test outcross cat(s) and they have the same DBE mutation as your current DBE lines, do not breed them together. Instead of improving eye colour you risk potentially serious problems in the descendants and risk ruining your breeding programme.

WHEN GENES INTERACT

The DBE mutations could be different mutations of the same region of a single gene, or mutations in different regions of a single gene, or mutations of different genes entirely. Genes come in paired alleles; each allele could have the same mutation or a different mutation (or no mutation at all). A cat might be heterozygous for 2 mutations at a single region AND heterozygous for mutations at a different region. It could be homozygous at both regions, or heterozygous at one region and homozygous at another region. A worst case scenario is that the gene is so badly damaged by the mutations that it cannot function, and no processes downstream of that gene can function properly (or at all) - resulting in deformity, disability or non-viable embryos. And that’s just for one gene! A cat might ALSO have a DBE mutation on a completely different gene, and the two different genes might also interact. Even if the genes are on a different chromosome, they might be involved in the same development pathway and again, their interaction could be disastrous.

This is why breeders should not mix multiple DBE mutations (mutations from different origins) into one breed. Breeders should avoid breeding different DBE breeds together unless both come from the same original cat and have the same mutation. A single "DBE Programme" that does not separate the genes is likely to end in disaster further down the line.

The "blue eyes is the next big thing" effect has become so widespread that a number of catteries now state they "do not work with the blue-eye genes" and that their blue-eyed cats are due to the colourpoint gene or the white gene.