Patent Application: US-31540308-A

Abstract:
the present invention relates to a method of diagnosing or detecting cone - rod dystrophy in a mammal , particularly in a canine species . in particular , the invention relates to methods involving the detection of a deletion in the nphp4 gene and associated biomarkers . the invention also provides primers , nucleic acid molecules , polypeptides , antibodies , as well as kits for use in such methods .

Description:
seq id no : 1 is the genomic sequence of the wild - type canis familiaris nphp4 gene . ( 5 dna : chromosome chromosome : broadd2 : 5 : 62820170 : 62935638 : 1 ) seq id no : 2 is part of the genomic sequence of canis familiaris nphp4 gene , from and including exon 4 to exon 6 . seq id no : 3 is the genomic sequence of the mutant canis familiaris nphp4 gene , with the 180 nucleotide deletion starting from and including nucleotide 19 of exon 5 . in the “ nnnn ” sequence , n can be any of the nucleotides a , c , g or t . ( in this sequence , all the nnnn - nucleotide stretches are in the introns ). ( 5 dna : chromosome chromosome : broadd2 : 5 : 62820170 : 62935638 : 1 ) seq id no : 4 is the cdna sequence of the wild - type canis familiaris nphp4 gene . seq id no : 5 is the cdna sequence of the mutant canis familiaris nphp4 gene with the deletion of exon 5 . seq id no : 6 is the full amino acid sequence of the wild - type canis familiaris nphp4 gene . seq id no : 7 is the full amino acid sequence of the mutant canis familiaris nphp4 polypeptide which results from the deletion of exon 5 . seq id no : 8 is the amino acid sequence encoded by wild - type nphp4 exon 5 . ( only ). seq id no : 9 is a fragment of seq id no : 3 which spans , but excludes , the exon 5 deletion . the contiguous sequence is present in mutant canis familiaris genomic dna . seq id no : 10 is the nucleotide sequence which encodes the amino acid sequence of seq id no : 6 . seq id no : 11 is the nucleotide sequence which encodes the amino acid sequence of seq id no : 7 . seq id no : 12 is the nucleotide sequence which encodes the amino acid sequence of seq id no : 8 . seq id nos : 13 - 94 are the primer sequences given in fig2 . seq id nos : 95 - 140 are the primer sequences given in fig3 . seq id nos : 141 - 204 are the primer sequences given in fig4 . seq id nos : 205 - 208 are the sequences given in fig8 . seq id no : 209 shows exon 5 and the start of intron 5 - 6 in the mutant canine nphp4 gene , as disclosed in the description . seq id no : 9 & lt ; 223 & gt ; exon 5 deletion is after nucleotide 132 seq id nos : 94 - 124 & lt ; 223 & gt ; primer used in amplifying exon of dab1 gene seq id nos : 125 - 136 & lt ; 223 & gt ; primer used in amplifying exon of ajap1 gene seq id nos : 137 - 140 & lt ; 223 & gt ; primer used in amplifying exon of vamp3 gene seq id nos : 141 - 204 & lt ; 223 & gt ; primer used in amplifying exon of nphp4 gene the invention will now be illustrated further by the following non - limiting examples . other features and advantages of the present invention will become apparent from the above detailed description . it should be understood , however , that the above detailed description and the following specific examples , while indicating preferred embodiments of the invention , are given by way of illustration only , since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description . day blindness was diagnosed in a wire haired standard dachshund and a purpose bred family of dogs confirmed that this most likely was a simple recessively inherited phenotype . detailed clinical descriptions of the affected family have been reported previously [ 52 - 53 ], establishing the disease as an early onset cone - rod dystrophy . all procedures used in this study were in accordance with the norwegian animal research authority (“ forsøksdyrutvalget ”) and adhered to the association for research in vision and opthalmology statement for the use of animals in opthalmic vision and research . blood samples were collected in edta - coated tubes from all the families segregating the disease and from two non - affected , unrelated dogs . genomic dna was isolated from the blood samples using dneasy tissue kit ( qiagen , gmbh , germany ). thirteen affected and thirteen sex - and age matched controls ( preferably full sibs ) were selected for whole genome snp analysis ( fig1 ). as some of the dogs were euthanized a number of samples from various tissues ( including retina and muscle ) were collected immediately post mortem and frozen on liquid nitrogen for rna analysis . total rna was isolated from tissue using rneasy fibrous tissue kit ( qiagen ). mrna for further use in the race protocol was isolated from the total rna using dynabeads oligodt particles ( invitrogen , carlsbad , calif ., u . s . a .). from the swhd family 13 pairs of cases and controls were chosen . where possible , mismatched full sibs ( n = 10 ) were used in the analysis to equally distribute population stratification effects among cases and controls . where a full sib pair was not available , mismatched half - sibs were used ( n = 2 pairs ). the founding sire ( case 201 ) and dam ( control 208 ) were included in the analysis . genomewide association analysis was undertaken using the affymetrix version 2 dog snp array “ platinum panel ” containing 49 663 snp markers ( affymetrix , santa clara , calif ., u . s . a .). snp genotypes were obtained by following the affymetrix protocol for the human 500k array protocol but with a smaller hybridization volume to allow for the smaller surface area of the canine array as described in [ 54 ]. detailed information on the arrays is available at http :// www . broad . mit . edu / mammals / dog / canine array /. the sibling transmission disequilibrium test ( sibtdt ) was carried out on all 49 663 snp using the “ dfam ” option in the analysis package plink [ 51 ]. data were filtered to exclude individual calls with brlmm calling probabilities lower than 0 . 01 , loci with low minor allele frequency (& lt ; 0 . 05 ), loci with low genotyping success rate ( failed calls & gt ; 0 . 1 ), and individual dogs with greater than 25 % of genotypes missing . after applying these filters , 24 798 snps and 25 dogs ( 13 cases and 12 controls ) remained in the analysis . the mean genotyping rate in the individual swhds included in the analysis was 96 %. genome - wide significance was ascertained through permutation of phenotypes ( n = 10 000 permutations ) over all 24 798 snps in the analysis ( emp2 in plink ). case - control association mapping using plink was applied to the same data with the same filtering parameters to test the efficacy of case - control association strategies in related samples . a region of association spanning from 57 . 6 and 72 . 8 mb was identified using the array analysis . primers for microsatellite analysis in the region of cfa5 ( 52 . 8 and 77 mb ), were identified by searching selected sequence windows of approximately 200 , 000 bp for microsatellite motifs . tetra - and dinucleotide repeat microsatellites with more than ten and nineteen repeats , respectively , were selected . primers for amplification of selected microsatellites were designed using primer3 ( http :// frodo . wi . mit . edu / cgi - bin / primer3 / primer3_www . cgi ) ( fig2 ). one primer in each pair was labelled with fluorescein . for genotyping , pcr was carried out as described above with 28 cycles . the sizes of the alleles were estimated with an automated genotyper ( abi prism ® 3100 genetic analyzer , applied biosystems , foster city , calif ., u . s . a .) with software for fragment analysis . chromosomal locations of the predicted canine orthologs of the candidate genes were determined by using the http :// genome . ucsc . edu web site . primers were designed in introns , using primer3 ( fig3 ), at a distance of approximately 100 - 200 bp from the intron / exon borders , to provide optimal pcr products for bi - directional sequencing . the primers for amplification on each of the canine nphp4 - exons were positioned in introns according to the described human sequence nm — 015102 ( fig4 ). the exon specific primers for amplification of cdna were based on the predicted canine sequence xm — 546745 . pcr amplification reactions were performed using 83 μm of each primer in a 15 μl reaction containing 1 . 5 μl dna prepared as described above , 1 × ammonium pcr buffer containing 1 . 5 mm mgcl2 , 83 μm each of datp , dctp , dgtp and dttp , and 0 . 4 units taq dna polymerase ( ambion , austin , tex ., u . s . a .). after an initial denaturation at 95 ° c . for 2 min and 30 sec , samples were amplified for 34 cycles at 95 ° c . for 30 sec , 58 ° c . for 40 sec and 72 ° c . for 50 sec , followed by a final extension of 72 ° c . for 5 min and 30 sec . all pcr products were bidirectionally sequenced using the pcr primers and the bigdye terminator v3 . 1 cycle sequencing kit ( applied biosystems ). amplification was done in a 10 μl reaction mixture containing 2 μl bigdye sequencing buffer , 1 . 5 μl bigdye sequencing enzyme , 1 μl pcr product , 0 . 5 μl sequencing primer ( 5 pmol / μl ) and 5 μl dh 2 o . the sequencing reaction was performed by cycle sequencing with the following protocol : initially 95 ° c . for 1 minute then 28 cycles each of 95 ° c . for 10 sec , 50 ° c . for 15 sec and 60 ° c . for 2 min . sequence reaction products were purified by ammonium acetate precipitation and sequenced on the abi3100 . one affected family member was compared to an unaffected english cocker spaniel ( ecs ) at the mrna level . total rna was isolated from 30 mg of muscle tissue using rneasy fibrous tissue kit ( qiagen , gmbh , germany ) following the manufacturers instructions . the rna was precipitated using standard procedures with ethanol , sodium acetate and glycogen . the rna was dissolved in 10 μl rnase free water and first strand cdna synthesis was performed using superscript ii rt following the manufacturer &# 39 ; s descriptions . the final cdna product was diluted 1 : 1 with rnase free water . exon specific primers were designed based on the predicted canine nphp4 sequence xm — 546745 . pcr with these primers were performed as described above with 1 μl cdna cdna as template . 3 ′- and 5 ′- race was performed to amplify the ends of the nphp4 transcript in both dogs . rna was isolated from 30 mg muscle tissue as described above , precipitated and dissolved in rnase free water . 5 ′- and 3 ′- end race was performed with generacer kit ( invitrogen , carlsbad , calif ., u . s . a .) and marathon cdna amplification kit ( clontech , mountain view , calif ., u . s . a . ), according to the manufacturer &# 39 ; s protocols . pcr and nested pcr was performed using kit specific 5 ′- and 3 ′- primers with reverse end forward gene specific primers and cloning of pcr products was performed using topo cloning kit . the inserts of the clones were amplified with m13 forward and reverse primer and sequenced with t3 and t7 primers . a significant association was detected to cfa5 using sib tdt analysis of ˜ 50 , 000 snps in 13 cases and 13 controls selected as discordant sib - pairs from a pedigree of standard wire - haired dachshund ( fig1 ). all samples were genotyped on the affymetrix v2 canine snp array . one control was excluded from further analysis based on low call rate (& lt ; 75 %). genome - wide significance ( prawtdt = 4 . 8e - 05 and pgenometdt = 0 . 00060 ) was achieved over the region cfa5 : 61 , 122 , 335 - 67 , 576 , 960 using a sibtdt analysis as part of the plink software [ 51 ]. homozygosity was detected in affected cases from 64 . 82 mb - 68 . 95 mb ( fig5 a and 5b ). case - control association identified the same region , but reported the highest association to a spurious snp in the “ shoulder ” of the linkage region ( p rawassoc = 1 . 3 × 10 − 5 , pgenomeassoc = 5 . 2 × 10 − 3 fig2 b ). in this region , allelic frequencies differed maximally between cases and controls , however one control dog was homozygous for the “ affected ” allele at the point of association ( chr5 : 69351798 ) and the foundation sire ( 201 ) was heterozygous in the near vicinity ( although not at this snp ). fine - mapping and haplotype analysis of microsatellites of the region between 52 . 8 mb to 77 . 0 mb revealed crossovers at 61 . 4 mb and 72 mb ( fig1 ). this region contains 130 genes that were examined to find possible candidate genes expressed in the retina . dab1 ( 55 . 3 mb , outside region ), ajap1 ( 62 . 0 mb ), vamp3 ( 64 . 4 mb ) and nphp4 ( 62 . 9 mb ) were identified as potential candidate genes . exon sequencing , including exon - intron boundaries , showed no mutations in the first three genes . pcr products from exon 5 of nphp4 showed a different size in unaffected ( 519 bp ) and affected ( 339 bp ) individuals and with both bands seen in carriers ( fig8 ). exon sequencing showed a deletion of 180 bp in exon 5 / intron 5 ( location 62 , 913 , 591 - 62 , 913 , 770 ) in the affected animal , where only the first 18 bases of exon 5 were retained ( fig6 ). screening of exon 5 in 72 unrelated swhd revealed 5 carriers , giving a gene frequency of the mutated gene of approximately 4 % in the swhd population . the exon screening also revealed 4 snps within exons and 4 snps were found in intronic sequence close to exons . all of the expressed snps give rise to amino acid changes , but all are present as polymorphisms in multiple breeds and are not associated to disease within the dachshunds ( fig7 ). no mutations were found in the exon / intron junctions . pcr products from human exon 5 of nphp4 using the primers in fig4 (“ humnphp4ex5 ”- f - primer and r - primer ) show the testing for the presence of a normal pcr product in a human . this shows that the method of the invention is easily adapted to the testing of human sampled ( fig8 c ). comparison of cdna from affected and unaffected dogs , revealed complete exon - skipping of exon 5 in the affected animals . translation of the 4819 bp long cdna sequence of the healthy dogs gives a 1429 amino acid ( aa ) protein , while translation of the cdna of the affected dog contains an early stop codon in exon 6 resulting in a short protein of 155 aa ( fig8 ). the race procedure of the 3 ′- and 5 ′- ends gave a complete mrna sequence , with an extra untranslated exon 1 of 73 bp located at 6312 bp upstream ( canfam2 . 0 62 , 935 , 566 - 62 , 935 , 638 ) of exon 2 . in addition exon 2 was found to have a 5 ′- utr of 35 bp and exon 29 a 3 ′- utr of 420 bp ( camfam2 . 0 62 , 819 , 750 - 62 , 820 , 169 ). the amplification of the transcript ends revealed no differences between the affected and the non - affected dogs . the amplified cdna also revealed a difference with the previously predicted transcript xm — 546745 . the cdna sequence , amplified in this study , contains an additional cag - triplet in front of exon 20 in all dogs . n . udar , et al ., identification of gucy2d gene mutations in cord5 families and evidence of incomplete penetrance , hum . mutat . 21 ( 2003 ) 170 - 171 . m . michaelides , et al ., a detailed study of the phenotype of an autosomal dominant cone - rod dystrophy ( cord7 ) associated with mutation in the gene for rim1 , br . j ophthalmol . 89 ( 2005 ) 198 - 206 . n . a . adams , a . awadein , h . s . toma , the retinal ciliopathies , ophthalmic genet . 28 ( 2007 ) 113 - 125 . r . allikmets , et al ., a photoreceptor cell - specific atp - binding transporter gene ( abcr ) is mutated in recessive stargardt macular dystrophy , nat . genet . 15 ( 1997 ) 236 - 246 . r . allikmets , et al ., mutation of the stargardt disease gene ( abcr ) in age - related macular degeneration , science 277 ( 1997 ) 1805 - 1807 . mollet g , silbermann f , delous m , salomon r , antignac c , saunier s . characterization of the nephrocystin / nephrocystin - 4 complex and subcellular localization of nephrocystin - 4 to primary cilia and centrosomes . hum mol genet . mar . 1 , 2005 ; 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