Candidate Gene Study: Investigating the Genetic Basis for Equine Deafness

Hello, scientific world. My name is Alecia Achimovich and I have come (in peace) to relate the going-on’s of Dr. Koren Lipsett’s lab. The research that I have taken up the reigns to conduct is an investigation of the underlying genetic basis for equine deafness in a particular pedigree of Spanish mustangs (yes, that pun was intended). The studied, affected individuals are also not pigmented. While these phenotypes are seemingly unrelated, there is a clear connection. It is known that during development, melanocytes migrate from the neural crest to the inner ear’s cochlea. Thus, if a mutation exists, causing dysfunction in the pigment producing cells, it is logical that these same cells would affect the sound wave sensing mechanisms of the inner ear.

 

Wayward Wind, the horse which sired two known deaf, non-pigmented foals with different mares.

Wayward Wind, the horse which sired two known deaf, non-pigmented foals with different mares.

Poco, one of the affected foals characterized by its deafness and lack of pigmentation.

Poco, one of the affected foals characterized by its deafness and lack of pigmentation.

This is where I come in. It is my job to find the mutated gene somewhere in the horse genome. However, sequencing the whole horse genome would not only be costly, but also produce a mountainous pile of data. Instead, we have identified genes which we believe may be the source of the problem in the horses. The genes KIT and EDNRB have been studied in the past and have not yielded a definitive SNP (single-nucleotide polymorphism) to account for the disease state. After re-sequencing these genes for 16 horses of which two are both deaf and completely white, this result was confirmed. To move forward, I have decided to study the genes MLANA and MITF.

Let's take a break to enjoy the beauty of gel electrophoresis (one of my regular tasks)

Let’s take a break to enjoy the beauty of gel electrophoresis (one of my regular tasks)

MLANA, also known as MART1 (Melanoma Antigen Recognized by T-cells), is known to form a complex with the protein product of pmel17 which is essential for pre-melanosome structure and successive differentiation (the determination of function for an uncommitted cell). As the acronym indicates, this gene is often targeted in immunotherapeutic attempts to free the body of infringing melanoma. Interestingly (in my opinion), the silencing or down regulation of this gene in recent studies caused dose dependent hearing loss. Regain of hearing to normal level after the discontinuation of the treatment was seen in over two thirds of patients. This fact supports the gene’s double importance to the aural sensory pathway as well as to the pigment production pathway.

The transcription factor MITF acts as a regulator in the pigment production pathway. It is downstream of the signal transduction cascade as started by KIT and regulates the rate of transcription of MLANA, among other genes. Mutated MITF has been observed in humans affected by Waardenburg Syndrome. The symptoms of this disease include hearing loss and irregular coloration. As this phenotype is congruent to that seen in the horse pedigree, the study of MITF was a logical next step.
In order for sequencing to be done, the mRNA sequence of each gene must be compared to the whole chromosomal DNA sequence. With this information, primers are designed to amplify the exons or coding sequence in the gene. The regions are amplified via PCR, purified, and sent to Yale to be sequenced by their facility. I then analyze the data, looking for polymorphisms across the horse population, hoping to find a mutation that is homozygous in the deaf, white horses and heterozygous in their parents.

Example of data received from Yale’s sequencing facilities.

Thus far, MLANA’s five exons have been fully sequenced and have not revealed any mutations. MITF has had 6 of its 10 exons sequenced. One mutation has been noted, but it does not seem to be linked to the disease-state.
In the future, if the mutation is not uncovered, the genes which are most commonly linked to auditory-pigment diseases will be studied.

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