(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0104424 A1
`Tuggle et al.
`(43) Pub. Date:
`Jun. 5, 2003
`
`US 20030104424A1
`
`(54) GENETIC MARKERS FOR IMPROVED
`DISEASE RESISTANCE IN ANIMALS (BPI)
`(76) Inventors: Christopher K. Tuggle, Ames, IA
`(US); Thomas J. Stabel, Ames, IA
`(US); Xianwei Shi, Kunming (CN);
`Martha A. Mellencamp, St. Joseph,
`MO (US)
`Correspondence Address:
`MCKEE, VOORHEES & SEASE, P.L.C.
`801 GRAND AVENUE
`SUTE 3200
`DES MOINES, IA 50309-2721 (US)
`(21) Appl. No.:
`10/161,968
`
`(22) Filed:
`
`May 31, 2002
`
`Related U.S. Application Data
`(60) Provisional application No. 60/294,668, filed on May
`31, 2001.
`Publication Classification
`(51) Int. Cl. ................................................... C12O 1/68
`(52) U.S. Cl. .................................................................. 435/6
`
`ABSTRACT
`(57)
`A method for determining improved disease resistance in
`animals is disclosed. The method assays for a novel genetic
`alleles of the BPI gene of the animal. The alleles are
`correlated with Superior disease resistance. Novel nucleotide
`Sequences, assays and primers are disclosed for the methods
`of the invention.
`
`Page 1
`
`Spectrum Ex. 1006
`IPR Petition - USP 10,000,795
`
`

`

`Patent Application Publication
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`US 2003/0104424 A1
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`I 0,1m81,
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`z 0.1m81, I
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`Page 2
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`Patent Application Publication
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`Jun. 5, 2003. Sheet 2 of 7
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`US 2003/0104424 A1
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`Page 3
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`

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`

`

`Patent Application Publication
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`Jun. 5, 2003 Sheet 4 of 7
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`US 2003/0104424 Al
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`Page 5
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`

`

`Patent Application Publication
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`Jun. 5, 2003 Sheet 5 of 7
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`US 2003/0104424 Al
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`

`

`Patent Application Publication
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`Jun. 5, 2003 Sheet 6 of 7
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`US 2003/0104424 Al
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`Patent Application Publication
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`Page 8
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`US 2003/0104424 A1
`
`Jun. 5, 2003
`
`GENETIC MARKERS FOR IMPROVED DISEASE
`RESISTANCE IN ANIMALS (BPI)
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`0001) This application claims benefit under 35 U.S.C.
`$119(e) of provisional application No. 60/294,668 filed May
`31, 2001.
`
`GRANT REFERENCE
`0002 Work for this invention was funded in part by ISU
`Grant No. 400-43-71-21-3337. The Government may have
`certain rights in this invention.
`
`FIELD OF THE INVENTION
`0003. This invention relates generally to the detection of
`genetic differences among animals. More particularly, the
`invention relates to genetic markers which have been iden
`tified in Several genes indicative of heritable phenotypes
`asSociated with improved traits, Such as disease resistance or
`performance. Methods and compositions for use of these
`markers in genotyping of animals and Selection are also
`disclosed.
`
`BACKGROUND OF THE INVENTION
`0004 Genetic differences exist among individual animals
`as well as among breeds which can be exploited by breeding
`techniques to achieve animals with desirable characteristics.
`For example, Chinese pig breeds are known for reaching
`puberty at an early age and for their large litter Size, while
`American breeds are known for their greater growth rates
`and leanness. Often, however, heritability for desired traits
`is low, and Standard breeding methods which Select indi
`viduals based upon phenotypic variations do not take fully
`into account genetic variability or complex gene interactions
`which exist.
`0005 There is a continuing need for an approach that
`deals with Selection for disease resistance at the cellular or
`DNA level. This method will provide the ability to geneti
`cally evaluate animals and to enable breeders to more
`accurately Select those animals which not only phenotypi
`cally express desirable traits but those which express favor
`able underlying genetic criteria. This has largely been
`accomplished to date by marker-assisted Selection.
`0006 RFLP analysis has been used by several groups to
`study pig DNA. Jung et al., Theor. Appl. Genet., 77:271-274
`(1989), incorporated herein by reference, discloses the use
`of RFLP techniques to show genetic variability between two
`pig breeds. Polymorphism was demonstrated for Swine
`leukocyte antigen (SLA) Class I genes in these breeds.
`Hoganson et al., Abstract for Annual Meeting of Midwestern
`Section of the American Society of Animal Science, Mar.
`26-28, 1990, incorporated herein by reference, reports on the
`polymorphism of Swine major histocompatibility complex
`(MHC) genes for Chinese pigs, also demonstrated by RFLP
`analysis. Jung et al. Animal Genetics, 26:79-91 (1989),
`incorporated herein by reference, reports on RFLP analysis
`of SLA Class I genes in certain boars. The authors state that
`the results Suggest that there may be an association between
`Swine SLA/MHC Class I genes and production and perfor
`mance traits. They further state that the use of SLA Class I
`
`restriction fragments, as genetic markers, may have potential
`in the future for improving pig growth performance.
`0007. The ability to follow a specific favorable genetic
`allele involves a novel and lengthy process of the identifi
`cation of a DNA molecular marker for a major effect gene.
`The marker may be linked to a Single gene with a major
`effect or linked to a number of genes with additive effects.
`DNA markers have Several advantages, Segregation is easy
`to measure and is unambiguous, and DNA markers are
`co-dominant, i.e., heterozygous and homozygous animals
`can be distinctively identified. Once a marker System is
`established, Selection decisions could be made very easily,
`Since DNA markers can be assayed any time after a tissue or
`blood sample can be collected from the individual infant
`animal, or even an embryo.
`0008. The use of genetic differences in receptor genes has
`become a valuable marker System for Selection. For
`example, U.S. Pat. Nos. 5,550,024 and 5,374,526, issued to
`Rothschild et al., disclose a polymorphism in the pig estro
`gen receptor gene which is associated with larger litter Size,
`the disclosure of which is incorporated herein by reference.
`U.S. Pat. No. 5,935,784 discloses polymorphic markers in
`the pig prolactin receptor gene which are associated with
`larger litter Size and overall reproductive efficiency, the
`disclosure of which is incorporated herein by reference.
`0009. The present invention provides a genetic markers,
`based upon the discovery of a polymorphisms in the porcine
`BPI gene, which correlate with resistance or susceptibility to
`pathogenic infection in pigs. This will permit genetic typing
`of pigs for their BPI allele and for determination of the
`relationship of specific RFLPs to resistance to infection. It
`will also permit the identification of individual males and
`females that carry the gene for improved resistance. Thus,
`the markers may be Selection tools in breeding programs to
`develop lines and breeds that produce litters containing more
`resistant offspring. Also disclosed are novel porcine BPIP
`genomic Sequences, as well as primerS for assays to identify
`the presence or absence of marker alleles.
`0010. According to the invention a polymorphism was
`identified in the BPI gene which is associated with the
`improved resistance to pathogenic infection.
`0011. It is an object of the invention to provide a method
`of Screening pigs to determine those more likely to produce
`offspring with improved pathogenic resistance, in the BPI
`gene.
`0012 Another object of the invention is to provide a
`method for identifying genetic markers for improved disease
`resistance.
`0013 A further object of the invention is to provide
`genetic markers for Selection and breeding to obtain pigs
`that will be expected to have a lower Susceptibility to
`infection than those without the favorable allele.
`0014. Yet another object of the invention is to provide a
`kit for evaluating a Sample of pig DNA for Specific genetic
`markers of disease resistance.
`0015 Additional objects and advantages of the invention
`will be set forth in part in the description that follows, and
`in part will be obvious from the description, or may be
`learned by the practice of the invention. The objects and
`
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`US 2003/0104424 A1
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`Jun. 5, 2003
`
`advantages of the invention will be attained by means of the
`instrumentality's and combinations particularly pointed out
`in the appended claims.
`SUMMARY OF THE INVENTION
`0016 To achieve the objects and in accordance with the
`purpose of the invention, as embodied and broadly described
`herein, the present invention provides a method for Screen
`ing animals to determine those more likely to have beneficial
`phenotypes or against those with deleterious phenotypes
`(Such as, or associated with, improved innate immunity,
`disease resistance or resistance to bacterial infection, as
`evidenced by bacterial count, lymphocyte count, neutrophil
`count, or monocyte count after challenge to identify animals
`which have Superior bacterial killing, or ability to stave off
`infection in a particular population, when bred, or raised or
`to Select against pigs which have alleles indicating unfavor
`able phenotypes. These traits may also be observed by
`assaying general indicia of overall health of the animal. AS
`used herein the term “biologically different disease resis
`tance” or “innate immunity” shall mean an ability to stave
`off infection that is superior to that which is observed when
`the favorable allele is not present as evidenced by indicia
`including but not limited to average lymphocyte count and
`percentage, monocyte count, neutrophil count and percent
`age and bacterial count after challenge or other measure
`ments of innate immunity as well as measurements of
`overall health of the animal Such as feed intake, weight gain
`and the like.
`0017 Thus, the present invention provides a method for
`Screening pigs to determine those more likely to have the
`improved trait of Superior disease resistance and/or those
`less likely to demonstrate those traits which method com
`prises the steps: 1) obtaining a sample of tissue or genomic
`DNA from an animal; and 2) analyzing the mRNA or
`genomic DNA obtained in 1) to determine which allele(s)
`is/are present. Briefly, the Sample of genetic material ana
`lyzed to determine the presence or absence of a particular
`allele that is correlated with a desirable trait, or one which
`is linked thereto.
`0.018 AS is well known to those of skill in the art, a
`variety of techniques may be utilized when comparing
`nucleic acid molecules for Sequence differences. These
`include by way of example, restriction fragment length
`polymorphism analysis, heteroduplex analysis, Single Strand
`conformation polymorphism analysis, denaturing gradient
`electrophoresis and temperature gradient electrophoresis.
`0019. In one embodiment, the polymorphism is a restric
`tion fragment length polymorphism and the assay comprises
`identifying the gene from isolated genetic material; exposing
`the gene to a restriction enzyme that yields restriction
`fragments of the gene of varying length; Separating the
`restriction fragments to form a restriction pattern, Such as by
`electrophoresis or HPLC Separation; and comparing the
`resulting restriction fragment pattern from an animal gene
`that is either known to have or not to have the desired
`marker. If an animal tests positive for the marker (or allele),
`Such animal can be considered for inclusion in the breeding
`program. If the animal does not test positive for the marker
`genotype, the animal can be culled from the group and
`otherwise used.
`0020. In a most preferred embodiment, the gene, or a
`fragment thereof, is isolated by the use of primers and DNA
`
`polymerase to amplify a Specific region of the gene which
`contains the polymorphism or a polymorphism linked
`thereto. Next, the amplified region is either directly Sepa
`rated or Sequenced or is digested with a restriction enzyme
`and fragments are again Separated. Visualization of the
`Separated fragments, or RFLP pattern, is by Simple Staining
`of the fragments, or by labeling the primers or the nucleoside
`triphosphates used in amplification.
`0021. In another embodiment, the invention comprises a
`method for identifying a genetic marker for disease resis
`tance traits, Such as bacterial counts, lymphocyte count,
`neutrophil count, or monocyte count after challenge. Male
`and female animals of the Same breed, breed croSS, or Similar
`genetic lineage are bred, and the disease resistance traits are
`determined. A polymorphism in the gene of each animal is
`identified and associated with the desired trait(s). Preferably,
`PCR-RFLP analysis is used to determine the polymorphism.
`0022. It is also possible to establish linkage between
`specific alleles of alternative DNA markers and alleles of
`DNA markers known to be associated with a particular gene
`(e.g., the BPI gene discussed herein) which have previously
`been shown to be associated with a particular trait. Thus, in
`the present Situation, taking a particular gene, it would be
`possible, at least in the Short term, to Select for pigs, or other
`animals, likely to have Superior disease resistance or ability
`to Stave off infection, or alternatively, against pigs likely to
`have inferior traits, indirectly, by Selecting for certain alleles
`of a particular gene associated with the marker alleles
`through the Selection of Specific linked alleles of alternative
`chromosome markers. Thus, in the present situation, taking
`the BPI gene, it would be possible, at least in the short term,
`to Select for pigs likely to produce disease resistance, or
`alternatively, against pigs likely to produce Susceptible lit
`ters indirectly, by selecting for certain alleles of the BPI
`asSociated marker through the Selection of Specific alleles of
`alternative markers located on the same chromosome BPI is.
`0023 The invention further comprises a kit for evaluating
`a Sample of DNA for the presence in genetic material of a
`desired genetic marker located in the gene indicative of a
`inheritable trait of disease resistance or ability to stave off
`infection. At a minimum, the kit is a container with one or
`more reagents that identify a polymorphism in the porcine
`BPI gene. Preferably, the reagent is a set of oligonucleotide
`primers capable of amplifying a fragment of the Selected
`gene that contains a polymorphism. Preferably, the kit
`further contains a restriction enzyme that cleaves the gene in
`at least one place, allowing for Separation of fragments and
`detection of polymorphic loci.
`0024.
`In another embodiment, the invention comprises a
`method for identifying a genetic marker for meat quality
`and/or growth in a particular population. Male and female
`pigs of the same breed or breed croSS or Similar genetic
`lineage are bred, and meat quality and/or growth produced
`by each pig is determined. A polymorphism in the BPI gene
`of each pig is identified and associated with the meat quality
`and/or growth. Preferably, RFLP analysis is used to deter
`mine the polymorphism.
`0025. In another embodiment, the invention comprises a
`method for identifying a genetic marker for meat quality
`and/or growth in any particular economic animal other than
`a pig. Based upon the highly conserved nature of this gene
`among different animals and the location of the polymor
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`US 2003/0104424 A1
`
`Jun. 5, 2003
`
`phisms within these highly conserved regions, is it expected
`that with no more than routine testing as described herein
`this marker can be applied to different animal species to
`Select for meat quality and/or growth based on the teachings
`herein. Male and female animals of the same breed or breed
`croSS or Similar genetic lineage are bred, and the meat
`quality and/or growth produced by each animal is deter
`mined and correlated. For other animals in which Sequences
`are available a BLAST comparison of Sequences may be
`used to ascertain whether the particular allele is analogous to
`the one disclosed herein. The analogous polymorphism will
`be present in other animals and in other closely related
`genes. The term “analogous polymorphism' shall be a
`polymorphism which is the same as any of those disclosed
`herein as determined by BLAST comparisons.
`0026. The following terms are used to describe the
`Sequence relationships between two or more nucleic acids or
`polynucleotides: (a) “reference Sequence', (b) “comparison
`window', (c) “Sequence identity”, (d) "percentage of
`Sequence identity”, and (e) “Substantial identity”.
`0027 (a) As used herein, “reference sequence” is a
`defined Sequence used as a basis for Sequence comparison.
`In this case the Reference BPI sequence. A reference
`Sequence may be a Subset or the entirety of a Specified
`Sequence; for example, as a Segment of a full-length cDNA
`or gene Sequence, or the complete cDNA or gene Sequence.
`0028 (b) As used herein, “comparison window” includes
`reference to a contiguous and Specified Segment of a poly
`nucleotide Sequence, wherein the polynucleotide sequence
`may be compared to a reference Sequence and wherein the
`portion of the polynucleotide Sequence in the comparison
`window may comprise additions or deletions (i.e., gaps)
`compared to the reference Sequence (which does not com
`prise additions or deletions) for optimal alignment of the two
`Sequences. Generally, the comparison window is at least 20
`contiguous nucleotides in length, and optionally can be 30,
`40, 50, 100, or longer. Those of skill in the art understand
`that to avoid a high Similarity to a reference Sequence due to
`inclusion of gaps in the polynucleotide Sequence, a gap
`penalty is typically introduced and is Subtracted from the
`number of matches.
`0029 Methods of alignment of sequences for comparison
`are well-known in the art. Optimal alignment of Sequences
`for comparison may be conducted by the local homology
`algorithm of Smith and Waterman, Adv. Appl. Math. 2:482
`(1981); by the homology alignment algorithm of Needleman
`and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for
`similarity method of Pearson and Lipman, Proc. Natl. Acad.
`Sci. 85:2444 (1988); by computerized implementations of
`these algorithms, including, but not limited to: CLUSTAL in
`the PC/Gene program by Intelligenetics, Mountain View,
`Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the
`Wisconsin Genetics Software Package, Genetics Computer
`Group (GCG), 575 Science Dr., Madison, Wis., USA; the
`CLUSTAL program is well described by Higgins and Sharp,
`Gene 73:237-244 (1988); Higgins and Sharp, CABIOS
`5:151-153 (1989); Corpet, et al., Nucleic Acids Research
`16:10881-90 (1988); Huang, et al., Computer Applications
`in the Biosciences 8:155-65 (1L992), and Pearson, et al.,
`Methods in Molecular Biology 24:307-331 (1994). The
`BLAST family of programs which can be used for database
`similarity searches includes: BLASTN for nucleotide query
`
`Sequences against nucleotide database Sequences; BLASTX
`for nucleotide query Sequences against protein database
`Sequences; BLASTP for protein query Sequences against
`protein database sequences; TBLASTN for protein query
`Sequences against nucleotide database Sequences, and
`TBLASTX for nucleotide query Sequences against nucle
`otide database Sequences. See, Current Protocols in Molecu
`lar Biology, Chapter 19, Ausubel, et al., Eds., Greene
`Publishing and Wiley-Interscience, New York (1995).
`0030 Unless otherwise stated, sequence identity/similar
`ity values provided herein refer to the value obtained using
`the BLAST 2.0 Suite of programs using default parameters.
`Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).
`Software for performing BLAST analyses is publicly avail
`able, e.g., through the National Center for Biotechnology
`Information (http://www.ncbi.nlm.nih.gov/).
`0031. This algorithm involves first identifying high scor
`ing sequence pairs (HSPs) by identifying short words of
`length W in the query Sequence, which either match or
`Satisfy Some positive-valued threshold Score T when aligned
`with a word of the same length in a database Sequence. T is
`referred to as the neighborhood word score threshold (Alts
`chul et al., Supra). These initial neighborhood word hits act
`as Seeds for initiating Searches to find longer HSPS contain
`ing them. The word hits are then extended in both directions
`along each Sequence for as far as the cumulative alignment
`Score can be increased. Cumulative Scores are calculated
`using, for nucleotide sequences, the parameters M (reward
`Score for a pair of matching residues; always >0) and N
`(penalty Score for mismatching residues; always <0). For
`amino acid Sequences, a Scoring matrix is used to calculate
`the cumulative Score. Extension of the word hits in each
`direction are halted when: the cumulative alignment Score
`falls off by the quantity X from its maximum achieved value;
`the cumulative Score goes to Zero or below, due to the
`accumulation of one or more negative-Scoring residue align
`ments; or the end of either sequence is reached. The BLAST
`algorithm parameters W, T, and X determine the sensitivity
`and speed of the alignment. The BLASTN program (for
`nucleotide Sequences) uses as defaults a wordlength (W) of
`11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and
`a comparison of both Strands. For amino acid Sequences, the
`BLASTP program uses as defaults a wordlength (W) of 3, an
`expectation (E) of 10, and the BLOSUM62 scoring matrix
`(see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA
`89:10915).
`0032.
`In addition to calculating percent Sequence identity,
`the BLAST algorithm also performs a statistical analysis of
`the similarity between two sequences (see, e.g., Karlin &
`Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)).
`One measure of similarity provided by the BLAST algo
`rithm is the smallest sum probability (P(N)), which provides
`an indication of the probability by which a match between
`two nucleotide or amino acid Sequences would occur by
`chance.
`0033 BLAST searches assume that proteins can be mod
`eled as random Sequences. However, many real proteins
`comprise regions of nonrandom Sequences which may be
`homopolymeric tracts, short-period repeats, or regions
`enriched in one or more amino acids. Such low-complexity
`regions may be aligned between unrelated proteins even
`though other regions of the protein are entirely dissimilar. A
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`
`number of low-complexity filter programs can be employed
`to reduce Such low-complexity alignments. For example, the
`SEG (Wooten and Federhen, Comput. Chem., 17:149-163
`(1993)) and XNU (Clayerie and States, Comput. Chem.,
`17:191-201 (1993)) low-complexity filters can be employed
`alone or in combination.
`0034) (c) As used herein, “sequence identity” or “iden
`tity” in the context of two nucleic acid or polypeptide
`Sequences includes reference to the residues in the two
`Sequences which are the Same when aligned for maximum
`correspondence over a Specified comparison window. When
`percentage of Sequence identity is used in reference to
`proteins it is recognized that residue positions which are not
`identical often differ by conservative amino acid substitu
`tions, where amino acid residues are Substituted for other
`amino acid residues with similar chemical properties (e.g.
`charge or hydrophobicity) and therefore do not change