Abstract:
The invention concerns an in vitro method of determining a genotype relating to hereditary nasal parakeratosis (HNPK) in a dog. According to the invention the presence or the absence of a genetic variation in the SUV39H2 gene sequence is indicative of said disorder. The invention also concerns polypeptide based methods for determining said disorder. Further, nucleic acids, polypeptides and antibodies usable in said method are disclosed.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority under 35 U.S.C. §119(a) to Finnish patent application No. 2012/5554 filed on May 25, 2012, which is incorporated by reference herein in its entirety. 
       FIELD OF THE INVENTION 
       [0002]    This invention relates to an in vitro method of diagnosing a genotype relating to hereditary nasal parakeratosis (HNPK) in a dog and materials usable in said method. Particularly this invention relates to genetic variation relating to HNPK in Labrador Retrievers. 
       BACKGROUND 
       [0003]    Hereditary nasal parakeratosis (HNPK) is a genodermatosis of Labrador Retrievers with a monogenic autosomal recessive inheritance (Pagé et al. 2003, Peters et al. 2003). HNPK-affected dogs develop crusts and fissuring of the nasal planum at a young age but are otherwise healthy. Histopathological changes consist of parakeratotic hyperkeratosis and an accumulation of proteinacious fluid (“serum lakes”) within the stratum corneum and the superficial stratum spinosum. HNPK currently cannot be cured, but the symptoms can be alleviated with symptomatic therapy. 
         [0004]    Due to monogenic autosomal recessive inheritance, a method to determine the genotype of a dog is of high importance. Heterozygous dogs are not affected and can phenotypically not be distinguished from homozygous free dogs. However, heterozygous dogs carry the mutant allele (“carriers”) and may transmit this disease to their offspring. 
         [0005]    At the moment breeders can identify carrier dogs only based on phenotype information collected from earlier generations and to eliminate the identified carriers from breeding programs, thereby reducing the frequency of genetic disease in a breed. Thus, there is a long felt need in the breeding of Labrador Retrievers for a genetic test that permits the identification of also healthy carriers of HNPK. Therefore, a genetic test method that can discriminate the three genotypes “free” (=homozygous wildtype), “carrier” (=heterozygous”), and “affected” (=homozygous mutant) is highly valuable for dog breeding as well as for veterinary medicine to confirm the diagnosis of suspicious cases. 
       SUMMARY OF THE INVENTION 
       [0006]    An object of this invention is to provide means for identifying breeding dogs, particularly Labrador Retrievers, that are at risk of transmitting HNPK genotype to its progeny. 
         [0007]    These and other objects are achieved by the present invention as described and claimed below. 
         [0008]    The first aspect of the invention is an in vitro method for determining the genotype relating to hereditary nasal parakeratosis (HNPK) in a dog, comprising determining the presence or the absence of a genetic variation in the SUV39H2 gene sequence in a biological sample from said dog, and indicating that said dog suffers or will suffer from said disorder or is at risk of transmitting said disorder to its progeny if said genetic variation is present in said biological sample. 
         [0009]    The second aspect of the invention is a polynucleotide. According to the invention the sequence of said polynucleotide is selected from the group consisting of:
   a) SEQ ID NO:6,   b) a sequence being at least 80% identical to the sequence SEQ ID NO: 2, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972;   c) a sequence encoding a polypeptide having SEQ ID NO: 1, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972 of SUV39H2 gene;   d) a sequence encoding a polypeptide having at least 80% identity to SEQ ID NO: 1, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972 of SUV39H2 gene; and   e) a sequence co complementary to any of the sequences of items (a) to (d).   
 
         [0015]    The third aspect of the invention is primer pair suitable for detecting a genotype relating to hereditary nasal parakeratosis (HNPK) in a dog, comprising a first and a second primer, each comprising a contiguous span of at least 14 nucleotides of the sequence SEQ ID NO: 2 or a sequence complementary thereto, wherein: 
         [0016]    a) said first primer hybridizes to a first DNA strand of the SUV39H2 gene; 
         [0017]    b) said second primer hybridizes to the strand complementary to said first DNA strand of the SUV39H2 gene; and 
         [0018]    c) the 3′ ends of said first and second primers are located on regions flanking the position 972 of SEQ ID NO: 2, or of nucleotide positions complementary thereto. 
         [0019]    The fourth aspect of the invention is a method for identifying a dog that carries a heterozygous or homozygous mutation relating to hereditary nasal parakeratosis (HNPK), comprising determining genetic variation in the SUV39H2 gene in the dog. 
         [0020]    The fifth aspect of the invention is a method for diagnosing genotype relating to hereditary nasal parakeratosis (HNPK), comprising hybridizing a nucleic acid from a dog with an isolated nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80% identical to said sequence, wherein nucleotide T at position 972 of the encoding gene has been replaced with G. 
         [0021]    The sixth aspect of the invention is an in vitro method of determining the risk of hereditary nasal parakeratosis (HNPK) in a dog or its progeny, comprising determining the presence or the absence of an epitope comprising an N324K mutation in a polypeptide EC 2.1.1.43 in a biological sample from said dog, wherein the presence of said epitope indicates that said dog suffers or will suffer from said disorder or is at risk of transmitting said disorder to its progeny. 
         [0022]    The seventh aspect of the invention is an antibody specifically recognizing the N324K epitope of dog EC 2.1.1.43 polypeptide. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0023]      FIG. 1 . Genome-wide association analysis (the coordinates in the figure refer to the previous CanFam 2.1 assembly and are not identical with the CanFam 3.1 coordinates in the text). (A) We identified a single, highly significant signal on chromosome 2. (B) The association signal was located in the first third of chromosome 2. (C) The black bars indicate homozygous segments in the 14 affected dogs. Homozygosity mapping defined an exact interval for the localization of the causative mutation. (D) Gene content of the critical interval as inferred from the corresponding human gene annotation. 
           [0024]      FIG. 2 . Sequence context of the causative mutation for HNPK. (A) In the upper half, the sequence is shown in the same orientation as the SUV39H2 gene. (B) This gene is in minus orientation on chromosome 2. Therefore, in the lower half of the figure the flanking sequence is also shown in the forward orientation, which corresponds to the coordinates of the CanFam 3 genome assembly. 
           [0025]      FIG. 3 . Schematic presentation of the resequencing strategy of an affected Labrador retriever. A 200 bp fragment library was prepared and 3 lanes of 2×100 bp sequences were collected on an illumina HiSeq2000 instrument.; In total 1 billion reads or 100 Gb raw data corresponding to 38.5 genome coverage were collected. The flow chart shows the data analysis strategy and illustrates how the experimental sequence reads were used to determine the variants between the affected Labrador Retriever and the Boxer reference genome. 
           [0026]      FIG. 4 . SUV39H2 gene, graph showing sequencing of “Control” (healthy), “carrier” and “HNPK affected” 
           [0027]      FIG. 5 . Sequence conservation of the SUV39H2 protein. The figure shows an amino acid alignment in the region of the critical N324K variant, which forms part of the catalytically active SET domain. The sequence of the orthologous SUV39H2 proteins from all vertebrates is identical in the region of the N-324 residue (upper part of the alignment). Even more distantly related paralogous H3K9 methyltransferases share an asparagine residue at this position (lower part of the alignment). The high sequence conservation supports the hypothesis that a mutation of asparagine at position 324 will affect the function of the SUV39H2 protein. 
           [0028]      FIG. 6  shows 27 annotated human genes located at region corresponding to 15 annotated genes in Labrador Retriever analyzed within this study. 
       
    
    
     DETAILED DESCRIPTION 
       [0029]    The invention is based on the surprising finding that genetic variation in SUV39H2 gene is related to hereditary nasal parakeratosis (HNPK) in a dog. The presence of said genetic variation indicates that said dog suffers or will suffer from said disorder or is at risk of transmitting said disorder to its progeny. This invention relates to an in vitro method of determining the genotype relating to hereditary nasal parakeratosis (HNPK) in a dog. The dog is preferably Labrador Retriever. 
         [0030]    The SUV39H2 gene encodes a histone methylase, which specifically methylates the lysine-9 residue of histone 3 (H3K9 methylase). Trimethylation of H3K9 is a hallmark of transcriptionally silenced heterochromatin. Cells may use this epigenetic modification to turn off specific genes, whose products are no longer needed, e.g. during cell differentiation. A lack of functionally active SUV39H2 may thus lead to delayed cell differentiation. The HNPK phenotype involves hyperproliferative keratinocytes that escape their normal differentiation as they move upwards from the basal membrane to the stratum corneum of the epidermis. SUV39H2 gene is highly conserved within vertebrates. 
         [0031]    Specifically, said genetic variation comprises a replacement of nucleotide T with G at position 972 of a gene encoding a polypeptide having SEQ ID NO: 1 or a polypeptide sequence at least 80%; preferably at least 85%, more preferably at least 90%, still more preferably at least 95% and most preferably at least 98% identical to said sequence. Said gene encoding the polypeptide having SEQ ID NO: 1 is SUV39H2 gene or a respective gene and shown as SEQ ID NO: 2. 
         [0032]    As used in the present context the term “identity” refers to the global identity between two sequences compared to each other from the first amino acid encoded by the corresponding gene to the last amino acid. The identity of the full-length sequences is measured by using Needleman-Wunsch global alignment program. The man skilled in the art is aware of the fact that results using Needleman-Wunsch algorithm are comparative only when aligning corresponding domains of the sequence. 
         [0033]    The presence of said genetic variation in both alleles of said SUV39H2 gene indicates that said dog suffers or will suffer from HNPK, see Table 1. 
         [0034]    The presence of said genetic variation in one of the two alleles of said SUV39H2 gene indicates that said dog is a healthy carrier of HNPK. 
         [0035]    The absence of said genetic variation indicates that said dog is a healthy non-carrier of HNPK. 
         [0036]    Before this invention it has not been possible to identify healthy carriers (heterozygous) before breeding; only dogs having the variation in both genes (both alleles of the SUV39H2 gene or respective gene) have been recognized based on phenotype. It is important to avoid accidental matings of healthy carriers as offspring from such matings bear a 25% risk of being HNPK affected. According to the animal welfare legislation in many countries, it is forbidden to breed animals, if one has to expect offspring that suffers from hereditary diseases. HNPK is a hereditary disease within this part of the animal welfare legislation. Therefore, breeders are obliged by law to avoid the mating of healthy HNPK carriers. 
         [0037]    There are numerous ways to accurately determine the SUV39H2:c.972T&gt;G genotype of a dog. The variation can be detected using e.g. PCR, real-time PCR, melting point analysis of double-stranded DNA, mass spectroscopy, direct DNA sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), high performance liquid chromatography (HPLC), single base primer extension, or other related methods. The critical information is that this variant really is the causative variant for the HNPK phenotype and that there is a 100% perfect correlation of the genotype at this variant with the HNPK phenotype (not considering any other hypothetical independent HNPK mutations). 
         [0038]    The varied polynucleotide is selected from the group consisting of:
   a) a sequence having SEQ ID NO: 2, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972; and   b) a sequence being at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95% and most preferably at least 98% identical to the sequence SEQ ID NO: 2, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972; and   c) a sequence encoding a polypeptide having SEQ ID NO: 1, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972 of SUV39H2 gene; and   d) a sequence encoding a polypeptide having at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95% and most preferably at least 98% identity to SEQ ID NO: 1, wherein said polynucleotide comprises replacement of nucleotide T with G at position 972 of SUV39H2 gene; and   e) a sequence complementary to any of the sequences of items (a) to (d).   
 
         [0044]    This invention is also directed to a pair of primers, comprising a first and a second primer and being suitable for detecting a genotype relating to hereditary nasal parakeratosis (HNPK) in a dog. Each primer comprises a contiguous span of at least 14 nucleotides of a sequence of SEQ ID NO: 2 or a sequence complementary thereto, wherein:
   a) said first primer hybridizes to a first DNA strand of the SUV39H2 gene;   b) said second primer hybridizes to the strand complementary to said first DNA strand of the SUV39H2 gene; and   c) the 3′ ends of said first and second primers are located on regions flanking the position 972 of SEQ ID NO: 2, or of nucleotide positions complementary thereto.   
 
         [0048]    Skilled man is able to adapt a suitable pair to possible slight variations within sequences of other breeds than Labrador Retriever used as an example in this study. In preferred embodiment the primers comprise at least 16, preferably 18 and most preferably at least 20 nucleotides. 
         [0049]    In one embodiment the pair of primers have a sequence of 5′-CTCCTCAACTATGGACAAATCG-3′ (SEQ ID NO: 3) and a sequence of 5′-TGCCACATCTTTCCATTCAG-3′ (SEQ ID NO: 4). 
         [0050]    A use of genetic variation in the SUV39H2 gene in a dog, preferably Labrador Retriever, as a marker for identifying a dog which carries heterozygous or homozygous mutation relating to nasal parakeratosis is within this invention. 
         [0051]    Also a use of an oligonucleotide encoding a polypeptide having SEQ ID NO: 1 or a sequence at least 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95% and most preferably at least 98% identical to said sequence wherein nucleotide T at position 972 of the encoding gene has been replaced with G in diagnosing genotype relating to HNPK is within the scope of this invention. Typically the encoding gene is SUV39H2 gene show as SEQ ID NO: 2. 
         [0052]    The hereditary nasal parakeratosis (HNPK) in a dog, preferably Labrador Retriever, can also be determined in vitro based on presence or the absence of mutation in a polypeptide EC 2.1.1.43 typically being encoded by SUV39H2 gene (or respective) in a biological sample from said dog. The specific mutation at polypeptide level is N324K. The presence of said epitope indicates that said dog suffers or will suffer from said disorder or is at risk of transmitting said disorder to its progeny. Preferably the polypeptide is defined by having sequence of SEQ D NO: 1. 
         [0053]    One of ordinary skill in the art is aware that the detection of the mutation is not restricted to any particular method, e.g. in mass spectroscopy, defining the polypeptide sequence or detection using antibodies. The antibody should specifically recognize the epitope. Examples of such antibodies are monoclonal antibodies raised against a fragment comprising said epitope or any purified antibodies. 
         [0054]    The invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the description above and in the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the invention. 
       EXAMPLES 
     Example 1 
     Identification of the Causative Mutation 
       [0055]    We performed a genome-wide association study using 13 HNPK affected Labrador Retrievers and 23 non-affected Labrador Retriever. We genotyped these dogs at 174,376 single nucleotide polymorphisms (SNPs) evenly distributed across the genome. The analysis of the genotyping data of genome-wide association study by plink (http://pngu.mgh.harvard.edui-purcell/plink/) indicated significant differences between the cases and the controls on chromosome 2 ( FIG. 2 ). We then further analyzed the affected dogs and found that all 13 affected dogs shared an identical homozygous segment on chromosome 2 spanning from 20,818,259-22,414,949 (CanFam 3.1 assembly). This indicates that all the HNPK affected are probably inbred to a single founder animal, which transmitted the mutant allele and spread it into the population. These results further indicate that the causative mutation for HNPK must be located in the ˜1.6 Mb interval indicated above. This region at the moment contains only 15 annotated genes (NCBI MapViewer, dog genome build 3.1) shown in  FIG. 2  whereas the corresponding syntenic human region contains 27 annotated genes shown in  FIG. 6 . 
         [0056]    We sequenced the entire genome of an HNPK affected Labrador 
         [0057]    Retriever on an illumina HiSeq2000 instrument at 38× coverage. We compared the obtained sequenced to the Boxer reference genome and identified ˜2,000 sequence variants in the critical interval of ˜1.6 Mb on chromosome 2 between the HNPK affected Labrador Retriever and the Boxer reference sequence. The procedure and algorithms used are schematically illustrated in  FIG. 3 . Comparison resulted in the identification of 1357 SNPs and 621 indels. Among these were a total of four predicted non-synonymous variants (Table 1). One of these was an artifact due to an error in the Boxer reference sequence. The other three non-synonymous variants were tested for association with the HNPK phenotype in additional dogs (Table 1). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Association of three non-synonymous variants with the HNPK phenotype 
               
             
          
           
               
                   
                 LA 
                 LA obligate 
                 LA unrelated 
                 various 
               
               
                 Genotype 
                 cases 
                 carrier 
                 controls 
                 breeds 
               
               
                   
               
             
          
           
               
                 ITGA8 c.363A &gt; G 
                   
                   
                   
                   
               
               
                 A/A 
                 0 
                 0 
                 1 
                 2 
               
               
                 A/G 
                 0 
                 1 
                 2 
                 2 
               
               
                 G/G 
                 15 
                 3 
                 5 
                 10 
               
               
                 RPP38 c.381A &gt; G 
               
               
                 A/A 
                 0 
                 0 
                 218 
                 40 
               
               
                 A/G 
                 0 
                 4 
                 57 
                 21 
               
               
                 G/G 
                 16 
                 0 
                 0 
                 16 
               
               
                 SUV39H2 c.972T &gt; G 
               
               
                 T/T 
                 0 
                 0 
                 297 
                 139 
               
               
                 T/G 
                 0 
                 4 
                 43 
                 0 
               
               
                 G/G 
                 20 
                 0 
                 0 
                 0 
               
             
          
           
               
                 NMT2 c 254delC 
                 error in reference sequence 
               
               
                   
               
             
          
         
       
     
         [0058]    For one of these variants (ITGA8:c.363A&gt;G), all three genotypes were observed in unrelated Labrador Retrievers without nose problems. Thus this variant can be excluded as being causative for HNPK. The second non-synonymous variant (RPP38:c.381A&gt;G) showed perfect association to HNPK in a large cohort of Labrador Retrievers. However, we found the mutant allele also in many dogs from other breeds and we also found dogs from other breeds that were homozygous for the mutant allele. As HNPK is assumed to be a disease specific to the Labrador Retriever breed, we consider it highly unlikely that this variant is causative for HNPK. Finally, the third of the three non-synonymous variants, SUV39H2:c.972T&gt;G, is perfectly associated with HNPK in a large cohort of more than 300 Labrador Retrievers. We tested also 139 healthy HNPK-free dogs from other breeds and found only the wildtype allele at this variant. Thus, the SUV39H2:c.972T&gt;G variant seems to occur exclusively in the Labrador Retriever breed, which indicates that this variant must have arisen after the separation of modern dog breeds. This allele distribution corresponds to the scenario of a relatively young mutation event in a Labrador founder animal, which led to the spread of HNPK in this breed. Therefore, the genetic data strongly suggest that SUV39H2:c.972T&gt;G really is the causative mutation. 
         [0059]    On the protein level the SUV39H2:c.972T&gt;G variant is predicted to lead to an exchange of asparagine to lysine at position 324 (p.N324K). This amino acid exchange affects an evolutionarily highly conserved position of the catalytically active so-called SET-domain of the SUV39H2 protein. In silico tools such as Sift and Polyphen strongly predict a functionally damaging effect of this variant. 
         [0060]    In conclusion, the SUV39H2:c.972T&gt;G variant leads to a loss of function of SUV39H2. A loss of function of SUV39H2 is expected to lead to changes in epigenetic silencing of chromatin and thus to cell differentiation defects. Thus, from a functional point of view it is plausible that SUV39H2:c.972T&gt;G really causes HNPK. 
       List of Sequences: 
       [0061]    SEQ ID NO: 1 wild type protein encoded by SUV39H2 
         [0062]    SEQ ID NO: 2 wild type genomic SUV39H2 DNA 
         [0063]    SEQ ID NO: 3 and 4; primers 
         [0064]    SEQ ID NO: 5 mutated protein encoded by SUV39H2 
         [0065]    SEQ ID NO: 6 mutated genomic SUV39H2 DNA 
         [0000]    Application of these Findings 
         [0066]    The gained knowledge can now be used to determine the HNPK genotype of a dog. This is illustrated in Table 2. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Interpretation of SUV39H2:c.972T &gt; G genotypes 
               
             
          
           
               
                   
                 Genotype 
                 Genotype 
               
               
                   
                 SUV39H2 c.972T &gt; G 
                 HNPK 
               
               
                   
                   
               
               
                   
                 T/T 
                 free = homozygous wildtype 
               
               
                   
                 T/G 
                 carrier = heterozygous 
               
               
                   
                 G/G 
                 affected = homozygous mutant 
               
               
                   
                   
               
             
          
         
       
     
       Technical Process of Determining the SUV39H2:c.972T&gt;G Genotype 
       [0067]    In our lab, we PCR amplified a fragment containing exon 4 and some flanking sequences of the SUV39H2 gene using a forward primer 5′-CTCCTCAACTATGGACAAATCG-3′ and a reverse primer 5′-TGCCACATCTTTCCATTCAG-3′. We subsequently sequenced the resulting ˜615 bp product using Sanger sequencing technology to determine the genotype at the SUV39H2:c.972T&gt;G variant. The details of the flanking sequence are depicted in  FIG. 2 . 
       REFERENCES 
       [0000]    
       
         Pagé N, Paradis M, Lapointe J M, Dunstan R W: Hereditary nasal parakeratosis in Labrador Retrievers.; Vet Dermatol. 2003 April; 14(2):103-110. 
         Peters J, Scott D W, Erb H N, Miller W H. :Hereditary nasal parakeratosis in Labrador retrievers: 11 new cases and a retrospective study on the presence of accumulations of serum (‘serum lakes’) in the epidermis of parakeratotic dermatoses and inflamed nasal plane of dogs., Vet Dermatol. 2003 August; 14(4):197-203. 
         PLINK (1.07), http://pngu.mgh.harvard.edu/purcell/plink/ 
         Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira M A R, Bender D, Mailer J, Sklar P, de Bakker P I W, Daly M J &amp; Sham P C (2007) PLINK: a toolset for whole-genome association and population-based linkage analysis. American Journal of Human Genetics, 81.