Patent Application: US-201114117195-A

Abstract:
this invention relates to methods for the detection of a bovine that is affected by or carrier of brachyspina . it is based on the identification of a 3 . 3 kb deletion in the bovine fanci gene that is shown to cause the brachyspina syndrome . the present invention provides methods and uses for determining whether a bovine is affected by or carrier of brachyspina by analyzing its genomic dna or its rna . the methods can be used to perform marker assisted selection or genomic selection for increased fertility in said bovine .

Description:
autozygosity - mapping positions the brachyspina locus in a 2 . 5 mb bta21 interval . between january 2008 and december 2009 , we obtained biological material from six holstein - friesian calves diagnosed with brachyspina . as the previously reported cases ( f . i . 4 ), the six affected animals traced back , on sire and dam side , to sweet haven tradition , a once popular artificial insemination ( ai ) holstein - friesian bull . genomic dna was extracted using standard procedures and genotyped using a previously described bovine 50k snp array ( 3 ). assuming that brachyspina is indeed inherited as a autosomal recessive defect and genetically homogeneous in holstein - friesian ( as suggested from pedigree analysis ), the six cases are predicted to be homozygous for a common haplotype encompassing the causative mutation . we performed autozygosity mapping using the assist program ( 3 ) and 15 healthy holstein - friesian bulls as controls , and identified a single genome - wide significant peak ( p & lt ; 0 . 001 ) on chromosome 21 ( bta21 ). the shared haplotype spans 2 . 46 mb ( btau4 . 0 : 20 , 132 , 767 - 22 , 588 , 403 ) encompassing 56 annotated genes ( fig1 ). targeted and genome - wide resequencing identifies the causative 3 . 3 kb brachyspina deletion in the fanci gene . several of the 56 genes in the interval are known to cause embryonic lethality when knocked out in the mouse . we amplified the corresponding open reading frames ( orf ) from genomic dna of cases and controls but did not find any obvious disruptive dna sequence variant ( dsv ). we then performed targeted sequencing of the entire 2 . 46 mb interval . a custom sequence capture array ( roche nimblegen ) was designed based on the bovine btau4 . 0 build , and used to enrich the corresponding sequences from total genomic dna of two affected individuals prior to paired - end sequencing ( 2 × 36 bp ) on an illumina gaiix instrument . resulting sequence traces were mapped to the btau4 . 0 build using mosaik ( http :// bioinformatics . bc . edu / marthlab ). in the targeted region , the coverage of non - repetitive bases averaged 90 . 45 ( range : 0 - 336 ) for the first sample , and 61 . 28 ( range : 0 - 189 ) for the second , to be compared with 0 . 01 ( range : 0 - 24 ) for the first and 0 . 01 ( range : 0 - 104 ) for the second sample outside the targeted region . the proportion of targeted non - repetitive bases with coverage & lt ; 10 was 0 . 12 for both samples . we used the gigabayes software ( gabor t . marth , boston college , http :// bioinformatics . bc . edu / marthlab ) to identify dsv and detected 2 , 368 snps and 572 indels for a total of 2 , 940 dsvs . one thousand thirty two of these corresponded to dsv previously reported in breeds other than holstein - friesian , and were therefore eliminated as candidate causative mutations . of the remaining 1 , 908 dsv only one was coding , causing a serine to glycine substitution in the l00516866 gene encoding a myosin light chain kinase - like protein . this dsv was not considered to be a credible candidate mutation underlying brachyspina . we then generated mate - pair libraries from self - ligated 4 . 8 kb (± 0 . 35 kb ) fragments of one brachyspina case and three unrelated , healthy controls and generated & lt ; 3 . 7 gb of sequence on a illumina gaiix instrument for each animal . resulting traces were mapped to the btau4 . 0 build using the burrows - wheeler aligner ( bwa )( 10 ), and alignments visualized with the integrative genomics viewer ( igv )( 11 ). analysis of the reads mapping to the 2 . 46 mb interval readily revealed a 3 . 3 kb deletion removing exons 25 - 27 of the 37 composing the fanci ( fanconi anemia complentation - group i ) gene . the deletion was apparent from a cluster of 27 mate - pairs mapping ˜ 8 kb apart on the btau4 . 0 build , and from the complete absence of reads mapping to the deleted segment for the brachyspina case , contrary to the three controls showing normal , uniform coverage in the region . retrospective analysis of the sequence traces captured from affected individuals confirmed the abrupt coverage drop at the exact same location . we designed a primer pair spanning the presumed deletion , allowing productive amplification of a 409 by product from genomic dna of affected and carrier animals but not of unrelated healthy controls from the same or other breeds . sequencing this amplicon defined the deletion breakpoints , confirming the 3 , 329 bp deletion ( fig2 a ). retrospective analysis of the sequence traces captured from affected individuals identified several reads bridging and confirming the breakpoint . conversely , primer pairs designed within the deletion did not yield any amplification from dna of affected individuals compared to healthy ones . assuming that the deletion of exons 25 to 27 results in the juxtaposition of exons 24 and 28 in the mrna , the 3 . 3 kb deletion is predicted to cause a frameshift at amino - acid position 877 substituting the 451 carboxyterminal amino - acids with a 26 - residue long illegitimate peptide . moreover , the ensuing stop codon in exon 28 is expected to cause nonsense mediated rna decay ( fig2 b ). with its homologue fancd2 , the fanci protein forms the id complex that localizes to damage - induced chromatin foci . fanci is essential for dna interstrand crosslink repair . like fancd2 , fanci is monoubiquitinated by the ubiquitin ligase fa core complex , and phosphorylated by the atm / atr kinase ( f . i . 12 ). missense , nonsense and splice - site variants in the fanci gene underlie ˜ 2 % of cases of fanconi anemia ( fa ) in human ( 12 , 13 ). fa patients exhibit heterogenous symptoms , including growth retardation , skeletal abnormalities , renal , cardiac , gastrointestinal and reproductive malformations ( reminiscent of bovine brachyspina ), as well as bone marrow failure , early onset of cancer and mortality at a young age . development of a diagnostic test directly interrogating the 3 . 3 kb fanci deletion and confirmation its causality . we developed a 5 ′ exonuclease genotyping assay that simultaneously interrogates the mutant and wild - type allele . the assay uses 5 ′- tgt tag ccc agc aga gga - 3 ′ ( seq id no : 3 ) and 5 ′- att ctg aat cca cta gat gtc - 3 ′ ( seq id no : 4 ) as wild - type pcr primer pair combined with 5 ′- gca cac acc tat ctt acg gta c - 3 ′ ( seq id no : 5 ) and 5 ′- ggg aga aga act gaa cag atg g - 3 ′ ( seq id no : 6 ) as mutant pcr primer pair , and 5 ′ hex - agt ccc agt gtg gct aag gag tga - 3 ′ iabkfq ( wild - type ) ( seq id no : 7 ) and 5 ′ fam - cca ttc cac / zen / ctt tct atc cgt gtc ct - 3 ′ iabkfq ( mutant ) ( seq id no : 8 ) as probes ( integrated dna technologies , leuven , belgium ). allelic discrimination reactions were carried out on an abi7900ht instrument ( applied biosystems , fosters city , calif .) for 40 cycles in 2 . 5 μl volume with a final concentration of 250 nm for each probe , 500 nm for wild - type primers , 350 nm for mutant primers , taqman universal pcr master mix 1x ( applied biosystems , fosters city , calif .) and 10 ng of genomic dna . typical results are illustrated in fig3 . as expected , all available brachyspina cases were shown by these test to be homozygous for the deletion . the deletion proved to be absent in a sample of 131 sires healthy animals representing ten breeds other than holstein . we then genotyped a random sample of 3 , 038 healthy holstein - friesian animals carriers of the deletions accounted for 7 . 4 % of the sample , while no animals were found to be homozygous . assuming hardy - weinberg equilibrium , the absence of homozygous animals in a sample of 3 , 038 individuals has probability & lt ; 5 %. this strongly suggests that homozygosity for the mutation is not compatible with normal health , i . e . that the 3 . 3 kb fanci deletion is causal . fig1 : schematic representation of the brachyspina locus . shown are : ( a ) the results of autozygosity mapping positioning the brachyspina locus on bovine chromosome 21 ; ( b ) the genotypes of six brachyspina cases and three healthy controls for 1 , 269 snp markers on chromosome 21 , showing the 2 . 46 mb region of autozygosity in black & amp ; white ; ( c ) the gene content of the 2 . 46 mb region ; ( d ) the structure of the fanci gene with indication of the position of the 3 . 3 kb brachyspina deletion . fig2 : schematic representation of the brachyspina mutation . ( a ) detailed view of the 3 . 3 kb brachyspina deletion deleting exons 25 , 26 and 27 of the bovine fanci gene . the sequences flanking the deletion breakpoints are given . ( b ) predicted effect of the 3 . 3 kb brachyspina deletion on the structure of the bovine fanci protein . fig3 : example of results obtained with the brachyspina 5 ′ exonuclease test . each animal is represented by a dot and the three clusters correspond to +/+, +/ d , and d / d animals respectively ; non template controls ( ntc ) are visualized as x . 1 . shuster d e , kehrli m e jr , ackermann m r , gilbert r o . ( 1992 ) identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in holstein cattle . proc natl acad sci usa . 89 : 9225 - 9229 . 2 . thomsen b , horn p , panitz f , bendixen e , petersen a h , holm l e , nielsen v h , agerholm j s , arnbjerg j , bendixen c . 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