Merle in Dogs (M - Locus)

With merle colouring, the dog has a pattern of darker and lighter areas that appears random. The eyes may be lighter or blue in colour. Merle is based on a SINE insertion in the PMEL gene; in the letter nomenclature of coat colours, “merle” corresponds to the M locus.
The inheritance pattern for the M allele is autosomal dominant with incomplete penetrance; if it is homozygous (M|M), serious health problems such as deafness and/or blindness can sometimes occur. Possible further organ problems are repeatedly discussed. 
The SINE mutation that causes merle is based on a highly variable DNA segment [poly(T) repeat]. The length of the poly(T) repeat can change with each cell division. The result is SINE mosaics in all tissues and germ cells. These mosaics cause both the individual, unpredictable marbling and the occurrence of merle offspring from parents with cryptic SINE sizes.

Correct genotypic testing and breeding planning are crucial to prevent the development of ‘double merle’ animals and associated health problems. 

Once the causative mutation had been identified (Clark et al., 2006), it became apparent that the occurrence of merle colouring does not always follow the rules that apply to typical autosomal dominant inheritance:
•    Animals with two copies of the wild-type PMEL gene (m/m) do not exhibit the merle phenotype. 
•    Animals with one or two copies of the gene (M/m, M/M) exhibit the merle phenotype. 
•    Homozygous animals (M/M) can have serious health problems.

The peculiarities became understandable when it was discovered that the SINE mutations vary in length:
- Shortened allels would not trigger merle coat markings (cryptic merle).
- With a strongly expanded SINE, the white colouring would be particularly intense (harlequin merle).

The mechanism that leads to SINE length variants via expansions and condensations also provided the explanation as to why parent animals without merle markings could produce offspring with merle markings.

The most differentiated allele nomenclature comes from Langewin et al. (2018) and provides for 7 different length classes: m (wild type, free), Mc, Mc+, Ma, Ma+, M and Mh

It is currently unclear whether matings, as presented in Wikipedia (source: https://de.wikipedia.org/wiki/Merle-Faktor), are actually without risk for the offspring. 
Current publications on this subject lead to different interpretations.

The transmembrane protein PMEL17 is produced in melanocytes and is responsible for the formation of the fibrillar matrix in early melanosomes (type I – II). This matrix serves the deposition and polymerisation of eumelanin.
The activity of PMEL is confirmed for all pigment-forming cells in the skin, hair follicles, iris, retina and inner ear. The often severe defects in certain double Merle animals indicate that PMEL17 is also necessary for the correct functioning of other cell types. 

3.1 Mutation: SINE-Insertion

The merle mutation is based on the integration of a canine-specific short interspersed nuclear element (SINE-Cf) in reverse orientation at the boundary between intron 10 and exon 11 of the PMEL gene. 
This insert contains: 
- a 15 bp target site repeat
- the head and body sequence of the SINE
- a poly(T) sequence (complementary to the transcribed poly(A) tail region
- and a cryptic splice acceptor sequence (cSAS).

3.2 Length-dependent exonisation

The length of the poly(T) tail acts as a switch for selecting the splice site:

• short poly(T) tails → normal splicing at the original acceptor site (oSAS) → correct PMEL protein.
• long poly(T) tails (> 55bp)  → displacement of the branch point distance → use of the cSAS → “exonisation” of the SINE fragment → abnormal PMEL protein with 52 additional amino acids.

The resulting protein does not form a stable fibrillar matrix, which leads to impaired eumelanin deposition and the characteristic lightening of the coat.

3.3 Formation of length variants – mosaic patterns

Replication slippage: the poly(A/T) sequence makes this section extremely susceptible to errors during cell division due to replication slippage during DNA replication. This leads to unpredictable lengthening or shortening of the SINE in the newly formed cells.

Somatic mosaics: Elongation or shortening can occur in individual melanocytes during embryonic development and in all cell divisions during the growth phase; depending on their SINE composition, the resulting cell clones produce either the unchanged basic colour or varying degrees of lightening, which manifest as marbled coat patterns.

Germ cell mosaics: If the same instability occurs during egg and sperm formation, the individual germ cells each have an individual SINE size. Depending on which germ cell is used for fertilisation, altered SINE lengths can be passed on to offspring. This occasionally results in merle puppies from parents who are themselves phenotypically non-merle.

Fragment length analysis can be used to examine an individuals' two PMEL genes for the presence and size of the SINE.
When evaluating the findings, it should be noted that the data collected only reflects the composition of the test material. The mosaics described appear regularly in the findings, with more than two M alleles of different sizes being found.

4.1 Length variants and categorization

By examining animals with varying degrees of merle, it was possible to determine the length variations found in different dog breeds (Langevin et al., 2018, Murphy et., 2018). This showed that the observed coat phenotype largely correlates with certain lengths, but there are also cases where the SINE length findings from the study material do not match the coat phenotypes. This confirms the unpredictability of the merle-causing mutation as a result of replication slippage and the resulting cell mosaics. 

4.2. Classification of variants

L* = Langevin et al. (2018)

Absolute fragment length determination provides an exact determination of SINE sizes. It is unclear whether pairings, as presented in Wikipedia (source: https://de.wikipedia.org/wiki/Merle-Faktor), are actually without risk for offspring.

Current publications lead to different interpretations:

Langevin et al. (2018) describe the short forms (Mc, Mc+, Ma) as functionally inactive and neutral to health. Accordingly, carriers of such ‘ineffective’ SINE are considered biologically safe for breeding and may be mated with true merle animals.

Varga et al. (2020) disagree with this general safety assessment: they point to expansions and contractions of the SINE in all tissues. This means that the SINE length in leukocytes (blood test material) does not necessarily correspond to that in skin, eye or germ cells. Thus, even an apparently ‘cryptic’ animal can carry merle-active cell lines and exhibit ‘expression double merle’ defects.

Brancalion et al. (2021) support the view of Varga et al. (2020): Here, the merle system is part of the group of pigment-associated genes with potential health risks. PMEL variants are considered, alongside MITF, KIT and PSMB7, to be causes of eye and hearing defects.
The necessity of veterinary breeding controls and genetic testing programmes to avoid double merle offspring is emphasised.

As a preventive measure, phenotypically merle-free mating candidates are tested to determine whether they carry an inactive (cryptic) SINE of any size.

Only matings in which one of the parents has the genotype m | m (merle-free) are permitted.

The rationale behind this regulation is the observation that dogs that do not have merle coat markings themselves and show a shortened SINE in blood tests may well have active SINE sizes in their germ cells (Vargas).

When testing for this breeding regime, 3 allele findings are sufficient:

• m = no SINE
• mc = cryptic SINE, potentially Merle
• M = merle

Dogs with manifest merle coat patterns do not need a merle test, as it is obvious that they have an effective SINE element.

When exonisation occurs (active SINE), the PMEL protein formed is defective. If heterozygosity (m | M) is present, the intact gene copy cannot provide sufficient functional PMEL protein, meaning that the eumelanin-based coat colour (black or brown) is not correctly expressed in the affected cells. For the individual cell, the effect is completely dominant. However, due to the mosaics, the overall appearance is mixed, hence the incomplete penetrance.

In dogs that have a copy without SINE in every cell due to the parent with the genotype (m | m), the merle coat pattern depends on the length variants that develop during embryonic development and through cell division during growth by replication slippage. 
It is not uncommon for animals with a blood test result of m | M (classic merle) to exhibit a degree of white colouring that is considered typical of a “harlequin” merle. However, the opposite is also observed: despite blood test results indicating a classic merle, the dogs exhibit no or only a slight degree of merle.

While Langevin uses a practical, breeding-friendly classification based on phenotype and length, Varga and Brancalion emphasise the important role of tissue mosaics and recommend that cryptic merle should not be classified as safe across the board.

Correct DNA-based trait assessment and breeding planning are crucial to reducing the risk of animals being born with health problems and to preventing potential suffering. The availability of the test allows breeders and breeding organisations to test the breeding animals in question and then use the results responsibly when considering matings.

1. L.A. Clark, J.M. Wahl, C.A. Rees, & K.E. Murphy, Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog, Proc. Natl. Acad. Sci. U.S.A. 103 (5) 1376-1381, https://doi.org/10.1073/pnas.0506940103 (2006).
2. Murphy, S.C., Evans, J.M., Tsai, K.L., Clark, L.A.: PMEL: correlating genotype with phenotype. Mob DNA 9:26, 2018. Pubmed reference: 30123327. DOI: 10.1186/s13100-018-0131-6.
3. Langevin M, Synkova H, Jancuskova T, Pekova S (2018) Merle phenotypes in dogs – SILV SINE insertions from Mc to Mh. PLoS ONE 13(9): e0198536. https://doi.org/10.1371/journal.pone.0198536
4. Varga, L., Lénárt, X., Zenke, P., Orbán, L., Hudák, P., Ninausz, N., Pelles, Z., Szőke, A.: Being Merle: The Molecular Genetic Background of the Canine Merle Mutation. Genes (Basel) 11:, 2020. Pubmed reference: 32560567. DOI: 10.3390/genes11060660.
5. Brancalion, L., Haase, B. and Wade, C.M. (2022), Canine coat pigmentation genetics: a review. Anim Genet, 53: 3-34. https://doi.org/10.1111/age.13154

Further Information is provide at: Online Mendelian Inheritance in Animals