Lavender Foal syndrome (LFS), also referred to as dilute lethal (Bowling 1996), lethal LFS (Schott & Petersen 2005) and coat colour dilution lethal (Fanelli 2005), is a condition only reported to affect Arabian foals (Bowling 1996; Fanelli 2005; Madigan 1997; Pascoe & Knottenbelt 1999) and is inherited in an autosomal recessive manner (Bowling 1996). Affected foals have an unusual dilute coat colour, demonstrate various neurological abnormalities, are not able to stand and nurse, and if the typical coat colour characteristic is overlooked may be incorrectly diagnosed as suffering from neonatal maladjustment syndrome (NMS), neonatal septicaemia or neonatal encephalopathy (Bowling 1996; Page et al. 2006). Post-mortem evaluations have failed to yield any macroscopic findings, suggesting a biochemical cause for LFS. The prevalence of LFS remains unknown (Fanelli 2005; Page et al. 2006). A recent study using SNP chip technology has allowed researchers to identify a candidate region of 1 Mb containing 216 candidate genes for the disease (Gabreski et al. 2009). Dilute mouse and rat mutants have defects in melanosome transport and a failure of their release into keratinocytes (Futaki et al. 2000; Takagishi & Murata 2006). Mice homozygous for the dilute mutation have dilute coat colour, show severe ataxia and opisthotonus and die within 3 weeks (Huang et al. 1998). These symptoms are very similar to those observed in LFS. The neurological aspect of the condition arises from aberrant transport of organelles in the neurons which in turn impairs synaptic regulation (Takagishi et al. 2007). The dilute colour observed is not because of abnormal pigment production but an abnormal dispersal of melanosomes within the hair shafts (Au & Huang 2002; Strobel et al. 1990). In man, Griscelli syndrome type I is an autosomal recessive genetic disorder associated with a mutation in MYO5A, which is characterized by pigmentary dilution with hypotonia, marked motor developmental delay and mental retardation (Pastural et al. 1997).
To identify the molecular defect underlying this disorder, we sequenced the coding region of the MYO5A gene in normal, affected and carrier animals. DNA was extracted from tissue and blood samples of four affected foals, their carrier sires and dams as well as four unaffected, non-carrier individuals using a phenol-chloroform DNA extraction protocol with ethanol washes. PCR amplification of the MYO5A coding region was performed using 12 sets of primers (Lavender1–12 in Table S1) designed to amplify 12 regions of coding sequence conserved between Mus musculus and Equus caballus. PCR amplification was performed for 35 cycles of 45 s at 95 °C, 1 min at 60 °C and 2 min at 72 °C with a final extension step of 8 min at 72 °C in 20 μl reaction volumes. PCR products were purified using the Invitek MSB Spin PCRapace Kit by Invisorb® and sequenced in 10 μl reactions using Bigdye v3.1 sequencing chemistry (Applied Biosystems) on the ABI 3130xl Genetic Analyzer (Applied Biosystems). Sequences of the affected and normal individuals were deposited in GenBank under the accession numbers HM063929 and HM063930 respectively.
Comparison of the nucleotide sequences between affected and normal individuals revealed only one sequence variation in the fragment amplified by primer set seven. A single-base deletion of cytosine at 4459 bp (c.4459delC) produced a frameshift that resulted in a premature stop codon (p.Arg1487AlafsX13). The substituted amino acid, Arginine, is conserved between human, mouse, rat and horse sequences, and the resulting truncation of almost half the protein tail (Fig. 1) is the causative mutation for the disorder. Direct sequencing of the region containing the deletion confirmed that affected and carrier individuals were homozygous and heterozygous for the deletion, respectively, while the deletion did not occur in normal individuals (Fig. 1). To confirm the specificity of the mutation, nine samples from individuals related to the four carrier foals as well as five unrelated control samples were genotyped. Fluorescently labelled primers were designed to amplify a 154 bp fragment flanking the deletion site (ACDF01 in Table S1). PCR amplification was performed for 35 cycles of 45 s at 95 °C, 45 s at 60 °C and 1 min at 72 °C with a final extension step of 8 min at 72 °C in 20 μl reaction volumes. PCR products were subjected to capillary electrophoresis using an ABI 3130xl Genetic Analyzer (Applied Biosystems). All affected individuals showed a single peak with a fragment length of 153 bp on strand software (Toonen & Hughes 2001; version 2.4.16 ), while normal individuals had a single peak at 154 bp. Heterozygous carriers had two characteristic peaks of 153 bp and 154 bp (Fig. 1).
Myosins are cargo binding proteins that move along actin filaments, amongst others, driven by ATP hydrolysis (Woolner & Bement 2009). Myosin-Va is expressed in the brain and skin (Takagishi & Murata 2006), where it functions in organelle transport and membrane trafficking (Reck-Peterson et al. 2000). Myosin-Va also plays a role in axonal and dendritic transport in neurons (Langford & Molyneaux 1998; Reck-Peterson et al. 2000). The myosin heavy chain consists of an N-terminal globular head that is conserved across the class V myosins, a neck region with an alpha-helical structure and a tail domain consisting of a helical coiled-coil interspersed with globular domains and ending in a C-terminal globular tail. The head of the protein contains sites for ATP hydrolysis and actin binding and is approximately 765 amino acids in length. The neck region of approximately 147 amino acids contains the calmodulin binding sites in the form of six IQ motifs (Sellers 2000). The alpha-helical tail is the site of dimerization, while its distal globular segment is responsible for cargo binding and protein localization (Langford & Molyneaux 1998). The globular tail of myosin-Va contains at least two separate binding sites with a high propensity for interacting with a wide range of different cargo molecules (Li & Nebenführ 2008). The c.4459delC mutation described here lies within the globular tail domain of the myosin-Va protein. The region where the c.4459delC mutation lies is within a well known deletion of a dilute mouse mutant, D20J, which is known to occur in all splice variants (Strobel et al. 1990).
Griscelli syndrome type 1 in man is associated with pigment dilution and neurological symptoms (Pastural et al. 1997), while the dilute lethal mouse and dilute-opisthotonus rat mutants exhibit dilute coat colours and intermittent opisthotonus (Futaki et al. 2000; Huang et al. 1998). These conditions, like LFS, are all associated with mutations in the MYO5A gene. Our present study shows that LFS is an autosomal recessive condition caused by a single-base deletion in the MYO5A gene on chromosome 1 of the horse.