Patent Application: US-201013512536-A

Abstract:
a method for detecting a genetic polymorphism associated with lavender foal syndrome or a predisposition thereto in a subject , the method including screening a genomic material sample from the subject for the presence of at least one polymorphism in a myo5a gene .

Description:
for the purposes of this specification , a “ polymorphism ” may include a change or difference between two related nucleic acids . a “ nucleotide polymorphism ” refers to a nucleotide which is different in one sequence when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence . a “ probe ” or “ molecular marker ” is an rna sequence ( s ) or dna sequence ( s ) or analogues , modified versions , or the complement of the sequences shown . this may include a “ genetic marker ”, which is a region on a genomic nucleic acid mapped by a molecular marker or probe . a “ probe ” is a composition labelled with a detectable label . a “ probe ” is typically used herein to identify a marker nucleic acid . a polynucleotide probe is usually a single - stranded nucleic acid sequence that can be used to identify complementary nucleic acid sequences , or may be a double - or higher order - stranded nucleic acid sequence which can be used to bind to , or associate with , a target sequence or area , generally following denaturing . the sequence of the polynucleotide probe may or may not be known . an rna probe may hybridize with its corresponding dna gene , or to a complementary rna , or to other type of nucleic acid molecules . as used herein the term “ functional discriminatory truncations ” mean nucleic acid sequences , modified nucleic acid sequences , or other nucleic acid variants which , although they are truncated forms of sequences presented herein or variants thereof , can still bind in a discriminatory manner to target gene or nucleic acid sequences described herein and forming part of the disclosed embodiments . the terms “ isolated ” or “ biologically pure ” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state . an “ amplified mixture ” of nucleic acids includes multiple copies of more than one ( and generally several ) nucleic acids . “ stringent hybridization conditions ” in the context of nucleic acid hybridization are sequence dependent and are different under different environmental parameters . generally , stringent conditions are selected to be about 5 ° c . lower than the thermal melting point ( t m ) for the specific sequence at a defined ionic strength and ph . the t m is the temperature ( under defined ionic strength and ph ) at which 50 % of the target sequence hybridizes to a perfectly matched probe . highly stringent conditions are selected to be equal to the t m point for a particular probe . an example of stringent wash conditions for , say , a southern blot of such nucleic acids is a 0 . 2 × ssc wash at 65 ° c . for 15 minutes . such a high stringency wash may be preceded by a low stringency wash to remove background probe signal . an example of a low stringency wash is 2 × ssc at 40 ° c . for 15 minutes . in general , a signal to noise ratio of 2 × ( or higher ) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization event . for highly specific hybridization strategies such as allele - specific hybridization , an allele - specific probe is usually hybridized to a marker nucleic acid ( e . g ., a genomic nucleic acid , an amplicon , or the like ) comprising a polymorphic nucleotide under highly stringent conditions . preliminary investigations had indicated that myo5a may be a possible candidate gene for lavender foal syndrome . sequencing of the coding region identified a single - base deletion in a conserved region of the tail domain . the deletion produces a truncated protein product through the insertion of a premature stop codon ( p . arg1487alafsx12 ). the deletion was confirmed as the causative mutation by genotyping affected , carrier and normal individuals . to identify the molecular defect underlying this disorder , the coding region of the myo5a gene in normal , affected and carrier animals was sequenced . 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 done using 12 sets of primers ( seq . id . nos . 1 to 24 , also referred to as lavender 1 to lavender 12 in table 1 ), 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 3130 × 1 genetic analyzer ( applied biosystems ). partial sequences of the myo5a gene of normal and affected individuals are attached as sequence 1 ( also referred to as seq . id . no . 27 ) and sequence 2 ( also referred to as seq . id . no . 28 ), respectively . partial sequence of myo5a gene from an affected horse with the c . 4459delc mutation which causes lavender foal syndrome . “-” indicates the single - base deletion of cytosine at 4459 bp ( c . 4459delc ) which produced a frameshift that resulted in a premature stop codon ( p . arg1487alafsx12 ). comparison of the nucleotide sequences between affected and normal individuals revealed only one sequence variation in the fragment amplified by primer set 7 (“ lavender7 ), i . e . seq . id . no . 13 and 14 . a single - base deletion of cytosine at 4459 bp ( c . 4459delc ) produced a frameshift that resulted in a premature stop codon ( p . arg1487alafsx12 ). the substituted amino acid , arginine , is conserved between human , mouse , rat and horse sequences and the resulting truncation of almost half the protein tail ( fig1 ) 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 ( fig1 ). in order to confirm the specificity of the mutation , 29 samples from individuals related to the four carrier foals as well as 5 unrelated control samples were genotyped . fluorescently labelled primers were designed to amplify a 154 bp fragment flanking the deletion site ( acdf01 in table 1 of supplementary data ). 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 3130x1 genetic analyzer ( applied biosystems ). affected individuals all showed a single peak with a fragment length of 153 bp on strand software ( university of california ; 2006 ; 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 ( fig1 ). myosins are cargo binding proteins that move along actin filaments , amongst others , driven by atp hydrolysis ( woolner & amp ; bement 2009 ). myosin va ( myova ) is expressed in the brain and skin ( takagishi & amp ; murata 2006 ) where it functions in organelle transport and membrane trafficking ( reck - peterson et al . 2000 ). diluted mouse and rat mutants have defects in melanosome transport and a failure of their release into keratinocytes ( futaki et al . 2000 ; takagishi & amp ; murata 2006 ). myova also plays a role in axonal and dendritic transport in neurons ( langford & amp ; molyneaux 1998 ; reck - peterson et al . 2000 ). in man , griscelli syndrome type i is an autosomal recessive genetic disorder associated with a mutation in myo5a which is characterised by pigmentary dilution with hypotonia , marked motor developmental delay and mental retardation ( pastural et al . 1997 ). 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 & amp ; molyneaux 1998 ). the globular tail of myova contains at least two separate binding sites with a high propensity for interacting with a wide range of different cargo molecules ( li & amp ; nebenführ 2008 ). alternative splicing in the coiled - coil of the tail region creates further cargo binding specificity in that different exons , with different binding domains , are expressed in specific tissues only . the brain isoform contains exon b with a binding domain for adaptors in the brain while in the skin exons d and f code for binding domains for melanophillin ( au & amp ; huang 2002 ). the c . 4459delc mutation described here lies within the globular tail domain of the myova protein . the region where the c . 4459delc mutation lies is within a deletion of a mouse mutant , d20j , which is known to occur in all splice variants ( strobel et al . 1990 ). mice homozygous for the dilute mutation have dilute coat colour , show severe ataxia and opisthotonus and die within three weeks ( huang et al . 1998 ). 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 due to abnormal pigment production but an abnormal dispersal of melanosomes within the hair shafts ( au & amp ; huang 2002 ; 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 ). the present disclosure provides evidence that lavender foal syndrome is an autosomal recessive condition caused by a single - base deletion in the myo5a gene on chromosome 1 of equines . surprisingly , the inventors uncovered not the well known dilute mouse deletion , d20j , which one would expect to find in the coding sequence examined because it is conserved between mus musculus and equus caballus , but the novel c . 4459delc polymorphism set out hereinbefore . the disclosed embodiments therefore provide a novel genetic marker associated with lavender foal syndrome . au , j . s . y . & amp ; huang , j . d . 2002 . a tissue - specific exon of myosin va is responsible for selective cargo binding in melanocytes . cell motility and the cytoskeleton , 53 , ( 2 ) 89 - 102 bowling , a . t . 1996 , “ medical genetics ,” in horse genetics , wallingford : cab international , pp . 105 - 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va gene . nature genetics , 16 , ( 3 ) 289 - 292 reck - peterson , s . l ., provance , d . w ., mooseker , m . s ., & amp ; mercer , j . a . 2000 . class v myosins . biochimica et biophysica acta ( bba )— molecular cell research , 1496 , ( 1 ) 36 - 51 schott ii , h . c . & amp ; petersen , a . d . 2005 . cutaneous markers of disorders affecting young horses . clinical techniques in equine practice , 4 , ( 4 ) 314 - 323 sellers , j . r . 2000 . myosins : a diverse superfamily . biochimica et biophysica acta , 1496 , ( 1 ) 3 strobel , m . c ., seperack , p . k ., copeland , n . g ., & amp ; jenkins , n . a . 1990 . molecular analysis of two mouse dilute locus deletion mutations : spontaneous dilute lethal20j and radiation - induced dilute prenatal lethal aa2 alleles . molecular and cellular biology , 10 , ( 2 ) 501 - 509 takagishi , y ., hashimoto , k ., kayahara , t ., watanabe , m ., otsuka , h ., mizoguchi , a ., kano , m ., & amp ; murata , y . 2007 . diminished climbing fiber innervation of purkinje cells in the cerebellum of myosin va mutant mice and rats . developmental neurobiology , 67 , ( 7 ) 909 - 923 takagishi , y . & amp ; murata , y . 2006 . myosin va mutation in rats is an animal model for the human hereditary neurological disease , griscelli syndrome type 1 . annals of the new york academy of science , 1086 , 66 - 80 woolner , s . & amp ; bement , w . m . 2009 . unconventional myosins acting unconventionally . trends in cell biology , 19 , ( 6 ) 245 - 252