WO2011067373A1 - Method for detecting polycythemia in a dog - Google Patents

Abstract

The present invention relates to a method for diagnosing primary polycythemia in dogs by means of detection of mutations in the JAK2 gene.

Description

 METHOD OF DETECTING POLYGLOBULA IN A DOG

The invention relates to a method for detecting primitive polycythemia in a canine mammal.

Polyglobulias are pathologies characterized by an increase in the mass of red blood cells, leading to an increase in the parameters of the red line in the circulating blood: hematocrit, hemoglobin and red blood cell concentration. This can lead to cardiovascular problems as well as thromboses.

 Faced with a subject presenting an increase of these blood parameters, it is advisable to pose the diagnosis as precise as possible, in order to adapt the management and to reduce the risk of complications.

 Primary polycythemia (also known as true polycythemia, polycythemia vera (PV) in humans, or polycythemia vera) is due to proliferation of precursor cells of the hematopoietic lineage. It is therefore a myeloproliferative disorder, which can be treated by a background chemotherapy with a myelotoxic antimetabolite which in particular decreases the synthesis of red blood cells (in particular hydroxyurea).

 Recently, the presence of a recurrent acquired mutation in the gene

JAK2 has been found in 90% of patients with PV (half of the other proven cases of PV in humans have another mutation in the same JAK2 gene).

 This mutation (V617F) modifies nucleotide 617, located in the pseudokinase domain of JAK2, leading to the substitution of a valine by a phenylalanine, which triggers the constitutive activation of the kinase responsible for polycythemia. Detection of the mutation has become a major diagnostic tool in humans.

Primary polycythemia (PP) is a rare disease in dogs, and probably underestimated, the symptoms being nonspecific and attributable to other diseases (such as weakness, polyuro-polydipsia, neurological signs related to hypoglycemia or hyperviscosity), and the non-existence to date of a diagnostic test. The diagnosis in the dog is made by elimination (other causes of the increase in the erythrocyte lineage can be eliminated), also using puncture or biopsy of bone marrow (these however, two methods are particularly invasive, costly and complex to implement).

 There is therefore a need to define a new diagnostic test for primary polycythemiaemia in dogs (and, in general, in the canine mammal), which is simpler, faster and more discriminating than the methods used for this purpose. day.

The invention thus relates to a method for the diagnosis of a primary polycythemia (proliferative syndrome of precursors of the erythrocyte line) in the canine mammal, in which mutations are sought in the sequence of the JAK2 gene of this mammal.

DESCRIPTION OF THE FIGURES

 Figure 1: Analysis of JAK2 exon 14 sequence in DNA extracted from peripheral blood DNA. A: Wild type sequence from blood cells of a control dog. B: mutated sequence obtained from blood cells of the dog No. 1. The arrows indicate the 3 positions of variation which increase the existence of a sequence heterogeneity in the blood cells.

C: Wild type sequence detected in an oral smear of # 1 dog (germline DNA). D: Amino acid sequences of the wild-type or mutant JAK2 human and canine proteins. The sequences indicated on A, B, C are the complementary sequences of the sequence SEQ ID No. 2.

 Figure 2: proliferation of BaF3 cells transfected with constructs expressing the JAK2 protein, in the presence (left) or absence (right) of Interleukin 3. Mutant 1 represents the JAK2 canine construct with the V617F mutation. Mutant 2 represents canine JAK2 construct with V617F / C618L mutations. WT represents the controls (Canine JAK2 construct without mutation) The results of 3 independent experiments are presented. Figure 3: Western blot analysis of STAT5 phosphorylation (on Tyr694) in total cell extracts of BaF3 cells transfected with constructs expressing canine JAK2. The cells were placed for 12 hours in DMEM containing 1% FBS in the presence or absence of IL3.

The present application makes it possible to provide a method for diagnosing a primary polycythemiae in a canine, and in particular a dog, which is rapid, and makes it possible to avoid the taking of bone marrow. This method is based on the detection of mutations acquired in the canine JAK2 gene. This gene has a strong homology with the human JAK2 gene, and in particular in the pseudokinase domain (Zhao et al., J Biol Chem 2005 Jun 17, 280 (24): 22788-92, see in particular Figure 1.C).

The invention thus relates to a method for in vitro diagnosis of a polycythemia in a canine, characterized in that it comprises the step of detecting the presence of a leucine at position 618 of the JAK2 protein sequence. said canid, represented by SEQ ID No. 1.

 In one embodiment, the method also includes the step of detecting the presence of phenylalanine at position 617 of SEQ ID NO: 1.

 In another embodiment, the method comprises only the step of detecting the presence of a phenylalanine at position 617 of SEQ ID NO: 1.

 This method is particularly suitable in dogs, SEQ ID No. 1 representing the protein sequence of the JAK2 protein in dogs.

In a preferred embodiment, this method is performed on a blood sample that has been taken from the canine.

 In a preferred embodiment, the canine has a chronic rise in hematocrit.

 In a preferred embodiment, the canine does not show an increase in its level of circulating EPO (erythropoietin).

It is understood that the sequence SEQ ID No. 1 represents a particular allele of the JAK2 gene in dogs. Other canids may have different alleles. Thus, it is possible that the sequence of the JAK2 gene of a canid other than the dog (or of a particular dog) may differ from the sequence SEQ ID No. 1 by a few nucleotides (presence of natural variations between alleles or between sequences close phylogenetic animals).

Consequently, the presence of leucine at position 618 and / or phenylalanine at position 617 (or the equivalent of these positions, determined as a function of the location of the pseudokinase domain in the sequence in question, and especially if take an isoform of JAK3 into consideration) sequence of the JAK2 protein of said canine is indicative of a primitive polycythemia.

Mutations on the protein can be detected via antibodies specifically recognizing these mutations, or affinity chromatography.

 Mutations-specific antibodies can be obtained by immunizing an animal with a mutant protein, and sorting antibodies from the resulting serum (removing antibodies cross-reacting with the non-mutated protein).

However, it is easier to detect mutations in the protein by looking for mutations existing at the genetic level.

 Thus, in a particular case, the F617 (or L618) mutation is due to the presence of a TTC codon at position 1849 (or a CTT codon at position 1852) of SEQ ID No. 2.

In a particular embodiment, the method comprises a step of amplifying the DNA present in a sample that has been taken from the canine.

 As mentioned above, the desired mutations are acquired, it is preferable that the sample is a blood sample. However, it can also be a bone marrow sample. This sample must contain cells for extraction and analysis of canine DNA.

The method may thus comprise a step of hybridization of the canid DNA with a probe specific for one or the other mutation.

 This probe can in particular be attached to a solid support.

In another embodiment, the method comprises a step of sequencing a portion of SEQ ID No. 2 containing nucleotides 1849 to 1854 of SEQ ID No. 2.

 Amplification can be performed with a single pair of primers, or carry out two rounds of PCR, the second amplification being carried out with primers located within the first amplified fragment.

A high or very high fidelity DNA polymerase is preferably used. Indeed, since it is sought the specific presence of defined nucleotides, it is important that the polymerase does not induce mutations in the amplified fragment. Such enzymes are commercially available. Mention may thus be enzymes Phusion ™ DNA Polymerases (Finnzymes Oy, Espoo, Finland) with an error rate of the order of 4.4 x 10 ^"7.

 When carrying out the amplification reaction, one can choose to amplify only the JAK2 gene (or a part of this gene), using a suitable primer cut, or to amplify, in the same reaction, different genes potentially present in the sample (including other susceptibility genes to other pathologies), using a plurality of different primers. Several pairs of primers are thus introduced into the reaction tube, each being specific for a gene that it is desired to amplify.

Detection of polymorphic nucleotides in the JAK2 gene is accomplished in any manner known in the art. Those skilled in the art know indeed multiple appropriate protocols that can be identified on search engines on the Internet. These include www.ipbs.fr/formation/biotech/mutations.pdf.

 It is thus possible to sequence the DNA fragment, in particular by the Sanger sequencing method using only three deoxynucleotides and a dideoxynucleotide and a primer (in particular a primer as mentioned above). The sequence products are then separated on polyacrylamide gel and the presence of the mutation is confirmed by observation of the size of the products.

 One can also do a primer extension reaction. In this case, the primer used is defined such that the first nucleotide to be incorporated after this primer corresponds to the polymorphic nucleotide.

 Mutations can also be detected by the HRM technique (High

Resolution Melt), or any other method of sequencing.

In a particular embodiment, said detection step comprises a step of hybridization of the DNA (preferentially amplified) with a probe specific for one or the other mutation, or both mutations. The hybridization of two DNA chains is due to the complementary base pairing A-T and G-C and is decreased during mismatch. The Tm of an oligonucleotide of 10 to 20 bases will therefore be lowered if there is a mismatch. To calculate the Tm of an oligonucleotide less than 30 nucleotides, we use the following relation: Tm = 2 (A + T) + 4 (G + C), in which A, T, G and C are respectively the number of each of these bases in the oligonucleotide. It is thus possible to use the "dot blot" method, by fixing a probe oligonucleotide carrying the mutation on a nitrocellulose membrane, and by applying the DNA present in the sample (or amplified DNA) under conditions allowing the specific hybridization of the polymorphism (the TM is calculated according to the composition of the selected probe oligonucleotide.

 In this case, the probe is attached to a solid support.

 In a particular embodiment, the hybridization is carried out on a DNA chip. A first spot contains an oligonucleotide exhibiting the JAK2 gene polymorphism at the codon positioned at 1849 of SEQ ID No. 2, a second spot contains an oligonucleotide exhibiting the JAK2 gene polymorphism at the codon set at 1852 of SEQ ID No. 2, and a third spot contains an oligonucleotide of a non-mutation-carrying JAK2 gene. The DNA is amplified and labeled with a fluorophore. It is then hybridized on the oligonucleotides attached to the chip. Fluorescence is observed if hybridization occurs.

 The presence of the two nucleotides mentioned above can also be detected by other methods. After amplification, the amplification product can be mixed with DNA that does not contain the mutations and hybridize these two oligonucleotides. The mismatches are then detected by enzymatic cleavage (in particular using Ce / I) or chemical cleavage. This detection method is well known and suitable enzymes are in particular described in EP 964929.

Example

 Materials and methods

 Blood samples were taken from five dogs suspected of having PP.

 These animals exhibited: chronic elevation of hematocrit, low EPO dosage (if available), chest x-ray and abdominal ultrasound to rule out chronic or neoplastic diseases that may be a cause of polycythemia.

DNA extraction

Genomic DNA was extracted from 200 μΙ of whole blood using the Qiagen Qiamp DNA Blood Kit Mini Kit according to the manufacturer's instructions. The quantity and quality of the DNA samples were evaluated with a Nanodrop spectrophotometer. PCR and sequencing

 Canine specific primers were designed based on the published canine genome (NC_006583 Canis lupus familiaris, NCBI database).

 The primers were designed in the intronic regions that frame exon 14 (encoding the pseudokine domain of JAK2 where the human mutation is found mainly).

 The PCR reaction was performed using the Ready Mix kit (Invitrogen Life Technologies, Paisley, UK). Each 50 μΙ contained 4 μΙ of the extracted DNA, 25 μΙ of Ready Mix solution, 1 μl of the reverse primer (5 'GTTTG GGC ATTTTAAC ATATAG C ATATT3', SEQ ID No. 3) and 1 μl of the primer. sense (5'ATGCATTTCTCTTACAGCAGACAGTATC3 \ SEQ ID NO: 4).

 The conditions for amplification were: initial denaturation at 95 ° C for 5 min, followed by 30 cycles of (95 ° C for 40s, 62 ° C for 45s and 72 ° C for 1.5 min), with a step d final elongation of 72 ° C for 3 min. The PCR products were purified using the QIAgen Purification Kit according to the manufacturer's instructions and sequenced.

Subcloning the PCR product

 In case of need the PCR products were subcloned into the plasmid pCR2.1 -TOPO (Invitrogen Life Technologies, Paisley, UK) and amplified in competent cells "One Shot chemically competent E. coli", using the kit. TOPO Cloning Systems (Invitrogen Life Technologies, Paisley, UK) and following the manufacturer's instructions. After overnight culturing in LB medium containing 50 μg / ml ampicillin, the plasmid DNA was isolated using the QIAgen Miniprep kit, following the manufacturer's instructions.

Cloning of canine JAK2 cDNA

 In order to express the JAK2 protein in BaF3 cells, complete cDNA was synthesized by GeneCust (Luxembourg) based on the published canine genome and cloned into pCDNA3 (Invitrogen Life Technologies, Paisley, UK).

 Site-directed mutagenesis made it possible to prepare two mutants:

 In Mutant 1, a single base change was introduced to generate a V617F mutant

 In mutant 2, 2 mutations were introduced to generate the mutant

V617F / C618L BaF3 cells in culture and transfection

 BaF3 / EpoR cells are a subclone of the Interleukin 3 (IL3) dependent BaF3 cell line that expresses the erythropoietin receptor.

 Cells were maintained in DMEM supplemented with 10% fetal calf serum and NL3.

 Transfection of BaF3 / EpoR cells with plasmids carrying the wild-type cDNA and mutant constructs was performed by nucleofection using the Amaxa Cell Line Kit Nucleofector Kit V (Lonza, France).

 48 hours after transfection, the cells were cultured in the standard medium supplemented with IL3 and geneticin for 4 weeks to establish stable lines.

Western blot

10 ⁷ cells were washed in cold PBS were lysed with 1 ml of 2X NuPage LDS Sample Buffer buffer (280mm TrisBase, 4% LDS, 20% glycerol, 1mM EDTA, 0.44mM SERVA Blue G250, 0.35mM). mM phenol red, pH 8.5) supplemented with 2X NuPage Reducing Agent (100 mM DTT) (Invitrogen, Carlsbad, CA, USA).

 Proteins were separated on 12% SDS-polyacrylamide electrophoresis gel (SDS-PAGE), and transferred to PVDF membranes (Immobilon-P Membrane Transfer, Millipore).

 The antibodies used are Anti-Phospho STAT5 Tyr694 and anti-STAT5 antibodies from Cell Signaling Technologies.

Results

 Direct sequencing of exon 14 of JAK2 was performed from all the blood DNA samples taken from 5 different dogs as described above.

 4 dogs exhibited a wild-type sequence relative to the healthy control sequence, and identical to the published sequence from NC_006583 (AD Ne represented by SEQ ID NO: 2). (Figure 1A),

 One animal had a mutated sequence in exon 14 (Figure 1B) (heterogeneity of sequences present in blood cells).

 Three bases were modified in positions 1849 (G → T), 1852 (T → C) and

1853 (G → T) (Figure 1B). These are mutations acquired because the sequencing of DNA obtained from the germ line (oral smear) showed the wild-type sequence only (Figure 1 C), which precludes the possibility of hereditary mutations.

 In order to elucidate whether the 3 mutations related to 3 mutations on 3 different alleles or to a mutant harboring the 3 mutations on a single allele, PCR products were subcloned and sequenced.

 While 12 clones showed the wild-type sequence, 3 clones contained the mutant sequence with three point mutations. No clones were observed with only one or two mutations.

 These results indicate that the three mutations are simultaneously present in an allele of the JAK2 gene. It is estimated that the frequency of mutated alleles in the sample is 20% for this animal.

 Analysis of the corresponding protein sequence showed that 2 amino acids were modified: valine at position 617 was replaced by phenylalanine and cysteine at position 618 was replaced by leucine (FIG. 1D). Thus, the V617F mutation is identical to the main JAK2 mutation found in humans and associated with polycythemia vera.

Expression of the mutant sequence in BaF3 confers NL3 independence. Mutations acquired in cancer cells often lead to constitutive activation of the signaling pathways involved in cell growth.

 The JAK2 V617F mutation detected in human patients with PV is responsible for the growth of erythrocyte progenitors independently of cytokines.

 To test whether the JAK2 mutation identified in the dog has the same effect, BaF3 cells containing the récepteur receptor (BaF3 Epo-R) were transfected with plasmids expressing either the wild-type (WT) cDNA or a

Mutant cDNA.

 In the absence of IL3, only clones carrying a mutant JAK2 gene are able to grow (FIG. 2), without it being possible to observe a significant difference between the clone carrying a single mutation and that carrying a single mutation. triple mutation.

Thus, the mutation of JAK2 in dogs induces a growth advantage in hematopoietic cells. JAK2 active mutated protein STAT5

 It has been determined whether the expression of the mutant JAK2 protein could activate intracellular signaling constitutively.

 One of the most important pathways activated downstream from JAK2 is the phosphorylation of STAT5.

 The transfected BaF3 cells were incubated in the presence or absence of IL3, and analysis of the extracted proteins revealed an increase in the level of phosphorylated STAT5 in cells expressing the mutated JAK2 protein.

 Thus, the double mutant JAK2 V617F / C618L (mutant 2) was able to induce phosphorylation of STAT5 in the absence of cytokines (Figure 3).

 Constitutive phosphorylation was also observed with the V617F mutation protein (mutant 1), although the signal appeared weaker than mutant 2 (Figure 3).

 These results confirm that the mutated canine JAK2 protein exhibits constitutive kinase activity leading to constitutive activation of the intracellular signaling pathways responsible for primary polycythemia.

Discussion

 These results show the existence of mutations acquired in the gene

JAK2 canine, in one in five animals suspected of primary polycythemia.

 These three mutations result in the V617F and C618L substitutions in the JH2 pseudokinase domain involved in self-inhibition of kinase activity.

 In humans the presence of double mutants at positions 617 and 618 (V617F-C618R) was found in 3 patients. However, the presence of 2 mutated amino acids is extremely rare in humans and the functional consequences of a double mutant compared to a single mutant have not been elucidated.

 In this case, it may be that double mutations are more common in dogs than in humans, or that chance has allowed the identification of a very rare mutant.

The presence of a single mutation V617F or both mutations V617F / C618L does not seem to bring any difference in terms of cellular signaling (STAT5 is constitutively phosphorylated in BaF3 cells transfected the single or double mutant). This constitutive activation of mutants JAK2 also leads to the independence of BaF3 transfected cells against IL3.

 Only one dog in the five dogs studied had a mutation in the region analogous to the mutated JAK2 region in humans.

 PP is poorly characterized in dogs, particularly because of the difficulty in making a definitive diagnosis and also because the disease can only be slightly symptomatic for months or years of evolution.

 It can be considered that another region of the JAK2 gene could be mutated in the 4 dogs found to be negative, as has been reported in humans for exon 12 of the JAK2 gene. Indeed, these rare mutations are often reported in patients with only polycythemia (ie platelet counts and normal white blood cells) which is the situation of all dogs tested in this study.

 Finally, the presence of additional acquired mutations in humans in the TET2 and ASXL1 genes has been reported.

 The question of the appearance of additional mutations in canine genes should be of interest.

 Indeed, the results reported here show that similar mutations are found in the JAK2 gene in humans and in dogs, in the presence of primitive polycythemia. This suggests a high level of similarity in the regulation of erythropoiesis between humans and animals.

 Thus, dogs with polycythemia and mutation in the JAK2 gene, as reported here, could be used as a new animal model to study the impact of mutations in the development of myeloproliferative syndromes, but also to test efficacy. new drugs.

Claims

claims 1. Method for the in vitro diagnosis of polycythemia in a canine, characterized in that it comprises the step of detecting the presence of a phenylalanine at position 617 of SEQ ID No. 1 (F617) and / or a leucine at position 618 (L618) of the sequence of the JAK2 protein of said canid, represented by SEQ ID No. 1. 2. Method according to claim 1, characterized in that it also comprises the step of detecting the presence of an F617 and an L618 in SEQ ID No. 1. 3. Method according to claim 1 or 2, characterized in that said canine is a dog. 4. Method according to one of claims 1 to 3, characterized in that the presence of F617 is due to the presence of a TTC codon in position 1849 and the presence of L618 is due to the presence of a codon and CTT at position 1852 of SEQ ID NO: 2. 5. Method according to one of claims 1 to 4, characterized in that it comprises a step of amplifying the DNA present in said sample. 6. Method according to one of claims 1 to 5, characterized in that it comprises a step of hybridization of the DNA with a probe specific for one or the other mutation. 7. Method according to claim 6, characterized in that said probe is attached to a solid support. 8. Method according to one of claims 1 to 5, characterized in that it comprises a step of sequencing a portion of SEQ ID No. 2 containing nucleotides 1849 to 1854 of SEQ ID No. 2.