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Canine Microphthalmia Transcription Factor (MITF) Gene and Merle Coat Color Genetics

Dr. Kate H. Eggleton, Clarion University of Pennsylvania

Introduction

The goal of this project is to determine if the Merle coat color homologous to, or linked to the Microphthalmia Transcription Factor (MITF) gene. In canine coat color genetics, black is dominant to red: Black = BB or Bb; Red = bb. Merle is codominant: Blue merle = B-Mm; Red merle = bbMm (fig. 1). The homozygous merle (MM) genotype is mostly white and linked to deafness & blindness (fig. 2).

The Jackson Hole Research Labs is home to the country’s largest colony of research mouse lines, called “JAX” Mice. One group of mice have been found to contain mutations in the Mitf gene (fig. 3 & 4). A ‘normal’ or ‘wild-type’ mouse has solid black coat color. Mice that are homozygous for the Mitf mutation (have two mutant copies of the gene) have a white coat and are usually deaf & blind. The gene exhibits codominant inheritance with gray or mottled coat color in heterozygote (one ‘normal’ copy and one mutant copy of the Mitf gene).

The Mitf gene has also been identified in Humans where it is called Waardenburg Syndrome type II (fig. 5). The Human MITF mutation is believed to be inherited in a dominant mode. Individuals with a single mutant allele are identified by their white ‘forelock’, and are often associated with hearing loss & loss of pigment in eyes.
Extensive research with the mouse lines has discovered at least 17 different Mitf mutations. The gene has been cloned & sequenced in both mouse and humans, and many of the properties of the protein coded for by the Mitf gene have been elucidated.

The Microphthalmia Transcription Factor gene product controls development of pigmented epithelia cells of the eye, osteoclasts & mast cells, and melanocytes. It may also control the transcription of tyrosinase, a gene required in the production of melanin. The MITF protein is a DNA binding protein of the basic helix-loop-helix leucine zipper (bHLH-Zip) family (fig.6).

Mutations in the Mitf gene can produce codominant, recessive, or dominant inheritance depending on which part of the protein is affected. Codominant inheritance is produced when the mutation is in DNA binding domain. In this case, the protein dimerizes normally - with protein from normal or mutant allele, but heterodimers can not bind DNA normally (fig.7). With recessive inheritance, the mutations are in the dimerization domains. The mutant protein can not dimerize and therefore cannot bind DNA efficiently (fig. 8). Dominant inheritance patterns have only been found in humans. Here, the DNA binding domain is affected. It has been suggested that human development is more sensitive to haploinsufficiency.

Could the merle coat color be the product of an Mitf gene mutation? We believe there is enough evidence to warrant testing this hypothesis. The inheritance pattern is similar to codominant mutants in mice, and there are several phenotypic similarities including the coat/hair color ‘dilutions’ and the ear & eye anomalies in homozygous condition.

Materials and Methods

Single Strand Conformational Polymorphisms (SSCP) and Heteroduplex (HD) analysis of amplified gene segments can effectively screen for single base mutations in nucleic acids. SSCP is based on the fact that single stranded (denatured) DNA molecules take on a sequence specific secondary structure (conformer) when transferred to non-denaturing conditions. Under the same conditions, some of the molecules will renature as a double helix. If there were two DNA sequences, differing only by a single base, heteroduplexes (double helices consisting of one of each sequence) as well as homoduplexes will form. The different conformers of single strands and the different duplexes can be detected due to differing mobilities in gel electrophoresis. used to identify linkage or homology of the merle gene with the mouse or human mitf gene.

DNA sample collection: DNA from normal and affected dogs were collected and isolated using “Catch-all” sample Collection Swabs and “BuccalAmp” DNA Extraction Kits from Epicentre, Inc. the FailSafe
PCR: The “FailSafe” PCR system was used to produce copies of exons that code for DNA binding domains. Primer sequences were based on exon sequences from human mitf clones.
SSCP and HD Analysis: PCR products were mixed with stop solution (1% xylene-cyanol, 1% bromphenol blue in formamide), denatured at 80oC for 5 min. then chilled on ice to obtain almost complete denaturation. 10u aliquots were immediately loaded on “Novex” 10 X 8 cm precast 20% TBE polyacrylamide gels (Invitrogen, Inc.). Gels were run at room temperature, 10 m amp/ gel, approximately two hours, and stained with ethidium bromide.

Results

PCR products using human template DNA and human control & Mitf exon primers as control have shown clear banding patterns on 20% PAGE gels. PCR using solid, merle and double merle canine DNA is now being tested with primers specific for exons in the mitf gene.