PCR-RFLP molecular confirmation of color dilution alopecia in dogs in Brazil

Abstract

Color dilution alopecia (CDA) is a dermatopathy observed exclusively in animals having a diluted coat color. In dogs, color dilution occurs as a result of a single-nucleotide variation (SNV) c.-22G > A in the melanophilin gene. We standardized a PCR–restriction-fragment length polymorphism (PCR-RFLP) technique to identify this mutation and determine its frequency in dogs in Brazil. The standardized PCR-RFLP technique could efficiently identify the SNV c.-22G > A in the melanophilin gene, with mutated allele frequencies of 0.1, 0.1, and 0.0875 in Dachshund, Miniature Pinscher, and Yorkshire Terrier breeds, respectively, with no statistical difference among the breeds (p = 0.252). The mutation was identified in 2 homozygous Dachshund dogs with alopecia, confirming the clinical characteristic of CDA. The standardization of a simpler and more accessible molecular technique for recognition of the SNV c.-22G > A in the melanophilin gene allows identification of heterozygous (phenotypically normal) dogs that can be excluded from reproduction, to avoid the birth of dogs with diluted coat color and consequently CDA.

Distribution of melanin in the hair cortex layer occurs through the participation of 3 proteins, encoded by the genes RAB27A, MYO5A, and MLPH.^5,6 Mutations in these genes are responsible for irregular arrangement of melanin granules, which results in the development of a diluted coat color. Thus, black-haired dogs become gray haired (also called blue); dogs with chocolate hair display a cream-colored phenotype (Isabella). ²

Dogs with diluted coat color are predisposed to the development of color dilution alopecia (CDA), which is a dermatologic disorder characterized by late hair loss, exclusively affecting the diluted coat. ⁷ CDA is described mainly in the Doberman Pinscher ⁷ ; however, other breeds, such as Dachshund, ² Yorkshire Terrier, ¹² Saluki, ⁸ Cane Corso, ⁹ and mixed-breed dogs ⁶ are also affected.

In dogs, a single-nucleotide variation (SNV) occurring as a result of replacement of the nucleotide guanine (G) by adenosine (A) at codon (c.)-22 (c.-22G > A) in exon 1 of the melanophilin (MLPH) gene is responsible for the dilution of hair color, ³ and is inherited as an autosomal recessive trait. Identification of heterozygous animals that are phenotypically normal (i.e., having undiluted coat color) is important in selecting breeding stock.

We aimed to standardize a PCR–restriction-fragment length polymorphism (PCR-RFLP) technique to identify dogs with the SNV c.-22G > A in the MLPH gene, and to determine its frequency in Dachshunds, Miniature Pinschers, and Yorkshire Terriers in Brazil. Informed consent was obtained from the owners for use of samples from their dogs. Our project was approved by the Ethics and Animal Use Commission of the Federal University of Mato Grosso do Sul, Brazil (protocol 822/2016).

For epidemiologic study of the MLPH mutation, 3 sample groups were formed: 40 Dachshunds, 40 Miniature Pinschers, and 40 Yorkshire Terriers. The purebred dogs were randomly selected from the routine care clinic of a veterinary hospital. The dogs were considered to be of their respective breed when they met the minimum standards determined by the Brazilian Confederation of Cynophilia (https://cbkc.org/cbkc), affiliated with the Fédération Cynologique Internationale.

All of the animals included in our study underwent clinical examination to exclude other causes of alopecia, such as dermatologic diseases of parasitic and endocrine origin. Genetic analysis of the animals was performed using blood samples collected by jugular venipuncture. At the time of collection, we recorded the phenotypic characteristics of the breed and the presence or absence of alopecia. The EDTA blood samples collected were stored frozen at −20°C until analyzed.

DNA was extracted from 350 μL of blood according to a published protocol. ¹ The DNA samples were subjected to 1% agarose gel electrophoresis, and bands were visualized with a UV transilluminator and gel documentation system (Bio-Rad) to evaluate the quality of the extracted samples.

For PCR analysis of the MLPH gene, we used primers described previously, ³ which are specific for the amplification of exon 1 of the MLPH gene. The forward (MLPH_157395_F: 5′-CCTTCCTTCCCCTGTAGGAC-3′) and reverse (MLPH_157706_R: 5′-GCCTAAAAT GAGCTCCCTGA-3′) primers amplified a fragment of 312 bp. Each sample was prepared in a final volume of 25 μL containing 10 μM of each primer (forward and reverse), 100 μg of DNA, and 12.5 μL of master mix (GoTaq Green; Promega), and nuclease-free water to make up the final reaction volume. The reaction conditions were as follows: 1 cycle at 95°C for 4 min; 35 cycles at 95°C for 30 s, 60°C for 20 s, and 72°C for 20 s; and 1 cycle at 72°C for 3 min. The amplified fragments were visualized using a UV transilluminator and gel documentation system after being subjected to 2% agarose gel electrophoresis.

For PCR-RFLP standardization, 2 DNA sequences from the fragment delineated by the primers, 1 from a non-carrier dog and 1 from a dog with the mutation, were subjected to in silico restriction analysis using Aligner v.8 (CodonCode). We used the restriction enzyme MspI, which generated different restriction patterns with the fewest number of fragments in carrier and non-carrier animals. The resultant bands were visualized by electrophoresis on 3% agarose gel. For validation of the PCR-RFLP, these results were compared to those obtained with the gene sequences of 8 DNA samples in our study.

The PCR products were purified and sequenced in both directions as described previously. ¹³ Purification was performed (Clean Sweep PCR reagent; Applied Biosystems) according to the manufacturer’s recommendations. The electropherograms were then analyzed using BioEdit v.7.2.5 (https://bioedit.software.informer.com), ⁴ and the consensus sequences generated were subjected to homology searches in GenBank.

Efficacy of the PCR-RFLP method was assessed using the kappa coefficient. The genotypic and gene frequencies obtained from alleles of the respective breeds were evaluated by relative frequency analysis. To verify if there was a statistical difference among the breeds, the chi-square test of adherence with determination of p-value was performed by Monte Carlo sampling (n = 100,000) in SAS v.9 (SAS Institute). ⁶

It was possible to identify the dogs that had the SNV c-22G > A mutation in the MLPH gene using the PCR-RFLP technique from whole blood samples. With the help of in silico analysis, we verified that the use of the restriction enzyme MspI allowed identification of dominant and recessive alleles, and heterozygous or homozygous animals on the basis of formation of fragments of different sizes. We verified that, based on the results of the first fragments obtained in our standardized PCR-RFLP technique, it was possible to distinguish the animals with the analyzed genetic mutation (Fig. 1).

Figure 1.

PCR-RFLP genotyping of SNV c-22G > A in the MLPH gene. The first column represents the molecular weight marker (50–500 bp). In the presence of the mutation in recessive homozygous (dd) animals, a band of 249 bp was formed. In heterozygous animals, 2 bands (249 bp and 237 bp; lane A) were observed, whereas in the absence of the mutation (DD), only 1 fragment was formed, represented by a single band of 237 bp (lanes B–F). Figures 2, 3. Two Dachshund dogs with generalized alopecia and the SNV c.-22G > A mutation in the MLPH gene, showing a homozygous phenotype. Note that alopecia affected the diluted colored coat exclusively.

Eight samples were selected to characterize the fragments of the MLPH gene amplified by Sanger sequencing, the gold standard technique for identifying mutations. The results were compared with those of the PCR-RFLP technique using the kappa coefficient, to analyze whether the results between the different techniques agreed. There was complete agreement (κ = 1.0) with the gold standard technique, thus allowing the application of this PCR method to identify animals with the analyzed genetic mutation. PCR-RFLP was performed on 40 animals of each of the described breeds to determine the frequency of the SNV c-22G > A mutation in the MLPH gene (Table 1).

Table 1.

Genotypic frequency of dominant homozygous (DD), heterozygous (Dd), and recessive homozygote (dd) and gene frequency of melanophilin gene “D” and “d” alleles in Dachshund, Miniature Pinscher, and Yorkshire Terrier dogs.

Breed	Number of animals	Genotypic frequency			Gene frequency
		DD	Dd	dd	D	d
Dachshund	40	0.85	0.1	0.05	0.9	0.1
Miniature Pinscher	40	0.8	0.2	0	0.9	0.1
Yorkshire Terrier	40	0.825	0.175	0	0.912	0.088
Total	120	0.833	0.15	0.05	0.904	0.096

At the time of blood collection, 5 dogs were alopecic (4 Dachshunds, 1 Doberman Pinscher); however, only 2 Dachshund dogs were homozygous for the mutation and had both the phenotypic and genotypic characteristics of CDA (Figs. 2, 3). Although the Dachshund breed was the only one in which we found recessive homozygosity, there was no statistical difference in genotypic frequency among the 3 analyzed breeds (p = 0.252).

We found that when the mutation was present in both alleles, 3 fragments (249, 43, and 20 bp) were formed using the MspI enzyme. In dogs in which the mutation was not present, the fragments were of 237, 43, 20, and 12 bp. Although the visualization of smaller bands is difficult, it is possible to identify the mutation by observing only the larger DNA fragments. The possibility of interpreting the data obtained using only agarose gel electrophoresis makes this technique promising for the identification of color dilution gene carriers.

Because color dilution is inherited as an autosomal recessive trait.^2,11 both parents of a litter of gray or Isabella pups must carry at least one recessive “d” allele for the pups to express color dilution. ⁷ Therefore, dogs bearing this allele should not be allowed to reproduce to avoid transmission of CDA to future generations. ⁷ The presence of homozygotes and heterozygotes to SNV c.-22G > A demonstrates the importance of monitoring this mutation for direct dog breeding in Dachshunds, Miniature Pinschers, and Yorkshire Terriers from Brazil. As an example of an extreme outcome of CDA, a dog with CDA developed squamous cell carcinoma throughout the dorsal trunk and head region. ¹⁰ Our molecular test can be utilized to avoid breeding animals carrying the mutation, thereby reducing the birth of animals predisposed to the development of CDA.