Patent Application
20240060133
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 350778_5T25.txt created on Feb. 18, 2022 and having a size of 40.8 kilobytes and is filed concurrently with the specification. The sequence listing comprised in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
Bleeding in animals is recognized as a significant health problem, especially in canines. In particular, several dog breeds can experience delayed bleeding in post-operative settings, including sighthound dog breeds such as racing greyhounds and Scottish deerhounds. This syndrome was first identified in retired racing greyhounds in 2007, indicating that bleeding disorders were one of the four most commonly reported causes of death in this breed, with a significant proportion of those deaths attributed to postoperative bleeding.
A subsequent prospective clinical study of greyhounds undergoing routine gonadectomy (ovariohysterectomy or castration) demonstrated unexpected postoperative bleeding in 26% of dogs, with unexplained bleeding observed 36 to 48 hours after the procedure. Signs of abnormal bleeding ranged from severe skin bruising around the surgical site to frank oozing of blood from the wound that was accompanied by a greater than 6% decrease in hematocrit. There was no evidence for bleeding at sites distant from the wound.
Although bleeding can spontaneously resolve without the need for transfusion of blood products a comparison of a range of measures of primary and secondary hemostasis in blood samples from affected and unaffected dogs failed to show any abnormality. However, whole blood thromboelastography suggests excessive fibrinolysis (hyperfibrinolysis), including poor clot strength, and lower plasma antiplasmin activity in affected dogs.
It has been suggested that the antifibrinolytic drug epsilon aminocaproic acid (EACA) could be utilized to prevent delayed postoperative bleeding. Over a 5-year period in 46 greyhounds that underwent limb amputation for osteosarcoma, 67% of greyhounds (4/6) experienced bleeding prior to introduction of prophylactic treatment. After introduction of prophylactic treatment with a fresh frozen plasma transfusion, 33% (5/15) of greyhounds experienced bleeding. After addition or substitution of this prophylactic treatment with EACA, the incidence of bleeding decreased to 16% (4/25) of greyhounds.
Despite these findings, the incidence of bleeding in dogs and the course of treatment—both prophylactically and therapeutically—remains erratic and unpredictable. This is especially evident given the unexplained occurrences in post-operative bleeding that occur several days after the surgical procedure takes place. Therefore, there exists a need for new methods to identify animals at risk for bleeding complications and treatment regimens to reduce the severity of the diagnosis. Accordingly, the present disclosure provides novel methods to identify animals via genetic testing and treat the animals at risk to prevent or reduce harmful bleeding episodes. In particular, the present disclosure provides determination of genetic mutations in the SERPINF2 gene and associated methods thereof.
The methods of the present disclosure provide several benefits compared to the current state of the art. The methods offer a simple genetic test to rapidly identify animals at risk for bleeding complications. Further, the methods provide a targeted treatment strategy for at-risk animals to mitigate a problem that, up to this point, has been unpredictable in nature. Further, identification of the described genetic mutation can also be beneficial in other methods as well, such as breeding decisions of animals to maximize the value of potential offspring.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific 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 this Detailed Description.
The following are definitions of terms that may be used in the present specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated. Additionally, it will be understood that any list of such candidates or alternatives is merely illustrative, not limiting, unless implicitly or explicitly understood or stated otherwise.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Abbreviations are used throughout this application, the following are abbreviations: EACA—epsilon aminocaproic acid; SNV—single nucleotide variant; AKC—American Kennel Club; NGA—National Greyhound Association; SDH—Scottish deerhound; PCR—polymerase chain reaction; O.R.—odds ratio; C.I.—confidence interval.
In addition, unless otherwise indicated, numbers expressing quantities of ingredients, constituents, reaction conditions and so forth used in the specification and claims are to be understood as being modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Various embodiments of the invention are described herein as follows. In an illustrative aspect, a method of treating bleeding in an animal provided. The method comprises the steps of determining if a genetic mutation in a SERPINF2 gene is present in the animal, and treating the animal with a veterinary composition if the genetic mutation in the SERPINF2 gene is present in the animal.
As used herein, a veterinary composition refers to any drug or formulation that can suitably be administered to an animal. For instance, veterinary compositions can include active ingredients approved for use in animals and/or active ingredients approved for human use and formulated or compounded for utilization in a non-human species. In some embodiments, the veterinary composition is obtained from a compounding pharmacy that formulates the veterinary composition for use in a non-human species using an FDA-approved drug or a base ingredient that is manufactured in an FDA-approved facility.
SERPINF2 (Serpin Family F Member 2) is known in the art as a protein coding gene. The gene encodes a member of the serpin family of serine protease inhibitors. The resultant protein is a major inhibitor of plasmin, which degrades fibrin and various other proteins. As described herein, genetic mutations of the SERPINF2 gene and/or its resultant protein can be utilized according to the present disclosure. As described herein, genetic mutations of the SERPINF2 gene and/or its resultant protein can be utilized according to the present disclosure.
In an embodiment, the step of treating is a prophylactic treatment. Generally, a prophylactic treatment can refer to treating an animal before a surgical procedure. For instance, a prophylactic treatment can refer to treating the animal at any point up to about 1 day prior to a surgical procedure, including immediately before such procedure.
In an embodiment, the step of treating is given before a trauma inducing incident of the animal. In an embodiment, the step of treating is given after a trauma inducing incident of the animal. For instance, a trauma inducing incident can refer to an injury to the animal or a surgical procedure. In an embodiment, the step of treating is given before whelping of the animal. In an embodiment, the step of treating is given after whelping of the animal.
In an embodiment, the bleeding in the animal is delayed bleeding. As used herein, a delayed bleeding can refer to bleeding from the animal that is evident at any time within 5 days after a surgical procedure. For instance, delayed bleeding can be evident within 6 hours after a surgical procedure, or within 8 hours after a surgical procedure, or within 12 hours after a surgical procedure, or within 1 day after a surgical procedure, or within 2 days after a surgical procedure, or within 3 days after a surgical procedure, or within 4 days after a surgical procedure, or within 5 days after a surgical procedure. In an embodiment, the delayed bleeding is associated with a surgery performed on the animal. In an embodiment, the bleeding in the animal is subsequent to a surgical procedure.
In an embodiment, the genetic mutation indicates that the animal is sensitive to experience delayed bleeding. In an embodiment, the genetic mutation is a single nucleotide variant. In an embodiment, the single nucleotide variant is a non-synonymous gene mutation. In an embodiment, the genetic mutation in the SERPINF2 gene is c.605 C>T (i.e., a cytosine→thymine mutation at nucleotide position 605). In an embodiment, the genetic mutation in the SERPINF2 gene is p.A202V (i.e., an alanine→valine mutation at amino acid position 202).
In an embodiment, the animal is a canine. In an embodiment, the canine is selected from the group consisting of a Greyhound, an Irish Wolfhound, a Basenji, an Italian Greyhound, a Galgo Espafiol, an Azawakh, a Scottish Deerhound, a Whippet, a Saluki, a Peruvian Inca Orchid, an English Bulldog, a Shetland Sheepdog, a Jack Russell Terrier, a French Bulldog, a Shiba Inu, a Boston Terrier, a Newfoundland, an Akita, a Golden Retriever, an Alaskan Malamute, an Australian Cattle Dog, a Boxer, a Border Collie, a Yorkshire Terrier, a Cairn Terrier, a Great Pyrenees, a Lhasa Apso, a Miniature Schnauzer, a German Shepherd, an American Staffordshire Terrier, a Pitbull, a Chesapeake Bay Retriever, a Beagle, a Collie, a Labrador Retriever, a Rottweiler, a Chow Chow, and a Mixed Breed.
In an embodiment, the canine is selected from the group consisting of a Scottish deerhound, greyhound, an Irish wolfhound, and a sighthound. In an embodiment, the canine is a Scottish deerhound. In an embodiment, the canine is a greyhound. In an embodiment, the canine is an Irish wolfhound. In an embodiment, the canine is a sighthound.
In an embodiment, the canine is a Basenji. In an embodiment, the canine is an Italian Greyhound. In an embodiment, the canine is a Galgo Espafiol. In an embodiment, the canine is an Azawakh. In an embodiment, the canine is a Whippet. In an embodiment, the canine is a Saluki. In an embodiment, the canine is a Peruvian Inca Orchid. In an embodiment, the canine is an English Bulldog. In an embodiment, the canine is a Shetland Sheepdog. In an embodiment, the canine is a Jack Russell Terrier. In an embodiment, the canine is a French Bulldog. In an embodiment, the canine is a Shiba Inu. In an embodiment, the canine is a Boston Terrier. In an embodiment, the canine is a Newfoundland. In an embodiment, the canine is an Akita. In an embodiment, the canine is a Golden Retriever. In an embodiment, the canine is an Alaskan Malamute. In an embodiment, the canine is an Australian Cattle Dog. In an embodiment, the canine is a Boxer. In an embodiment, the canine is a Border Collie. In an embodiment, the canine is a Yorkshire Terrier. In an embodiment, the canine is a Cairn Terrier. In an embodiment, the canine is a Great Pyrenees. In an embodiment, the canine is a Lhasa Apso. In an embodiment, the canine is a Miniature Schnauzer. In an embodiment, the canine is a German Shepherd. In an embodiment, the canine is an American Staffordshire Terrier. In an embodiment, the canine is a Pitbull. In an embodiment, the canine is a Chesapeake Bay Retriever. In an embodiment, the canine is a Beagle. In an embodiment, the canine is a Collie. In an embodiment, the canine is a Labrador Retriever. In an embodiment, the canine is a Rottweiler. In an embodiment, the canine is a Chow Chow. In an embodiment, the canine is a Mixed Breed.
In an embodiment, the veterinary composition comprises an active ingredient comprising a plasmin inhibitor. As used herein, the term “veterinary acceptable carrier” refers to any agents which do not cause an intolerable side effect and which allow the active ingredients in the veterinary composition to retain their pharmacological activities. A veterinary acceptable carrier includes excipients, emulsifiers, solubilizers, surfactants, buffers, preservatives, and/or other additives which may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, pharmacodynamics, reduce adverse side effect or provide other advantages for veterinary use. In an embodiment, the veterinary acceptable carrier is selected from the group consisting of selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof. In an embodiment, the veterinary composition comprises a further active ingredient.
In an embodiment, the veterinary composition is a unit dose. In an embodiment, the veterinary composition is a single unit dose. As used herein, the term “unit dose” is a discrete amount of the veterinary composition comprising a predetermined amount of one or more components. The amount of the components is generally equal to the dosage of the components which would be administered to an animal or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
A plasmin inhibitor refers to a composition that provides inhibition of plasmin, in both in vitro and in vivo settings. For instance, a plasmin inhibitor can bind to the same sites on plasmin to which alpha-2 antiplasmin binds. In an embodiment, the plasmin inhibitor is administered to the animal at a therapeutically effective dose, for example a dose at which plasmin is inhibited.
In an embodiment, the plasmin inhibitor is administered to the animal via an injection. In an embodiment, the injection is an intravenous injection. In an embodiment, the injection is an intradermal injection. In an embodiment, the injection is a subcutaneous injection. In an embodiment, the injection is an intramuscular injection. In an embodiment, the plasmin inhibitor is administered to the animal orally.
In an embodiment, the plasmin inhibitor is administered to the animal every 24 hours. In an embodiment, the plasmin inhibitor is administered to the animal every 12 hours. In an embodiment, the plasmin inhibitor is administered to the animal every 8 hours. In an embodiment, the plasmin inhibitor is administered to the animal every 6 hours. In an embodiment, the plasmin inhibitor is administered to the animal every 6 to 8 hours. In an embodiment, the plasmin inhibitor is administered to the animal every 8 to 12 hours.
In an embodiment, the plasmin inhibitor is selected from the group consisting of an aminocaproic acid, a tranexamic acid, a yunnan baiyao, and any combination thereof. In an embodiment, the plasmin inhibitor is an aminocaproic acid. In an embodiment, the aminocaproic acid is administered at a dose of 25 mg/kg to 50 mg/kg. In an embodiment, the aminocaproic acid is administered at a dose of 25-50 mg/kg every 6 to 8 hours Aminocaproic acid is also known as ε-aminocaproic acid, ε-Ahx, and 6-aminohexanoic acid and has a structure of:
In an embodiment, the plasmin inhibitor is a tranexamic acid. In an embodiment, the tranexamic acid is administered at a dose of 10 mg/kg to 15 mg/kg. In an embodiment, the tranexamic acid is administered at a dose of 10 mg/kg to 15 mg/kg every 8 to 12 hours. Tranexamic acid is also known as TXA and has a structure of:
In an embodiment, the plasmin inhibitor is a yunnan baiyao. In an embodiment, the yunnan baiyao is administered at a dose of 12.5 mg/kg to 25 mg/kg. In an embodiment, the yunnan baiyao is administered at a dose of 12.5 mg/kg to 25 mg/kg every 12 hours. Yunnan baiyao is a traditional Chinese herbal remedy and can comprise several compounds.
In an embodiment, the animal is administered a second therapeutic agent.
In a further illustrative aspect, a method for detecting sensitivity to bleeding in an animal is provided. The method comprises the step of determining if a genetic mutation in a SERPINF2 gene is present in the animal, wherein the presence of the genetic mutation in the SERPINF2 gene indicates that the animal is sensitive to bleeding. The previously described embodiments of the method of treating bleeding in an animal are applicable to the method for detecting sensitivity to bleeding in an animal described herein.
In an embodiment, the method further comprises the step of making a breeding decision based on the presence of the genetic mutation in the SERPINF2 gene. For instance, if a genetic mutation in the SERPINF2 gene is detected, an affirmative breeding decision based on the presence of the genetic mutation could be made. Alternatively, if a genetic mutation in the SERPINF2 gene is not detected, an affirmative breeding decision based on the presence of the genetic mutation could be made.
In a further illustrative aspect, method for making a breeding decision for an animal is provided. The method comprises the steps of determining if a genetic mutation in a SERPINF2 gene is present in the animal, wherein the presence of the genetic mutation in the SERPINF2 gene indicates that the animal is sensitive to bleeding, and making the breeding decision based on the presence of the genetic mutation in the SERPINF2 gene. The previously described embodiments of the method of treating bleeding in an animal are applicable to the method for making a breeding decision for an animal described herein.
The following numbered embodiments are contemplated and are non-limiting:
For the instant example, Scottish deerhounds with delayed postoperative bleeding and matched controls were identified through a survey distributed to members of the Scottish Deerhound Club of America. A subsection of this survey requested information regarding the surgical history of the dogs, occurrence of postoperative bleeding complications, and perioperative administration of antifibrinolytic drugs (EACA or tranexamic acid). A diagnosis of delayed postoperative bleeding, defined as unexpected bleeding from a surgical wound starting 1 to 4 days after the procedure, was based on review of the owner's description of the event, the veterinary medical record (if available), and therapeutic response to administration of antifibrinolytic drugs (if used). Dogs were excluded if the bleeding event occurred on the same day of surgery, or after more than 4 days following surgery.
Control dogs were included if they had undergone a surgical procedure where the owners reported no evidence for delayed postoperative bleeding. They were excluded as controls if antifibrinolytic drugs had been administered prophylactically. DNA samples from cases and controls were obtained either directly by cheek swab collection from the dogs by the owners or were archived samples obtained from the Canine Health Information Center (CHIC) DNA repository, or PennGen Laboratories (University of Pennsylvania).
Seven candidate genes involved in regulating fibrinolysis and clot strength (see
Gene regions targeted for sequencing included all exons as well as the 5′- and 3′-untranslated regions, based on NCBI Annotation Release 105 of the available public canine genome assembly. For the 10 DNA samples that were sequenced, the median probe depth for individual samples ranged from 25 to 521, with a median value of 53 among all samples. Reads were mapped to the canine reference genome (CanFam3.1) using Bowtie for Illumina (Galaxy Version 1.1.2). Mapped reads were then visualized with Golden Helix GenomeBrowse version 2.1.2 using the NCBI Refseq 104 Genes annotation and the NCBI dbSNP version 146 variant annotation. Sequence variants in the coding region were identified by comparing the sequences of mapped reads for each DNA sample with the annotated gene reference sequence. PolyPhen2 was used to evaluate the effect of identified missense SNVs on protein function.
A custom allele discrimination assay (Applied Biosystems TaqMan SNP Genotyping Assay, Thermo Fisher Scientific, Waltham MA) was developed to genotype DNA samples for the SERPINF2 c.605 C>T mutation. The primers and probes were 5′-ACG CTG CGG AGG TTA GAG-3′ (forward primer; SEQ ID NO:7), 5′-CCC AGG TCC TGG CAA AGG-3′ (reverse primer; SEQ ID NO:8), 5′-CCA GAG TCT GCA TGC AG-3′ (C-allele probe labelled with VIC dye; SEQ ID NO:9), and 5′-CCA GAG TCT ACA TGC AG-3′ (T-allele probe labelled with FAM dye; SEQ ID NO:10).
Assays were performed according to the manufacturer's directions using a real-time PCR instrument (CFX96 Touch, Bio-Rad, Hercules, CA). Assay accuracy was verified by genotyping the same case/control Scottish Deerhound DNA samples that had been sequenced using an independent gene capture approach (see above). F7 c.407 G>A mutation genotype was determined by Sanger sequencing of PCR product generated using primers 5′-ATC AAA CCT CAG CGG GGC TGG-3′ (Pri-1291; SEQ ID NO: 11) and 5′-GGG CTT GTT TCC GAG CGG G-3′ (Pri-1292; SEQ ID NO: 12).
Healthy NGA-registered greyhounds (N=19) aged between 1 and 10 years old were recruited from eastern Washington state and northern Idaho. Health was ascertained by review of the dog's medical history and a physical exam. Studied dogs included 6 neutered males, one intact male, and 12 spayed females aged between 2 and 9 years (average 4.9 years). Blood samples (3 mL) were collected by a single venipuncture from the cephalic or saphenous veins, immediately transferred to tubes containing sodium citrate anticoagulant and centrifuged. The plasma layer was removed and stored at −80° C. until analysis. Cheek swab DNA samples were also collected from each dog and used to determine SERPINF2 c.605 C>T genotype.
Plasmin Inhibition Assay
A microplate-based colorimetric kinetic assay was developed and validated to measure the potency of inhibition of canine plasmin by plasma based on a previously published method. Briefly, 2 μL of canine plasma diluted to 50 μL in phosphate-buffered saline (PBS) was added on ice to 96-well UV clear nonbinding plates (Cat. #655901, Greiner Bio-One, Monroe, NC). Then, 50 μL of canine plasmin (Cat. #DPLM, Molecular Innovation, Novi, MI) at 100 nM concentration in 50 μL PBS was added, mixed by pipetting, and incubated at 37° C. for 5 min. To this, 100 μL of prewarmed plasmin substrate (D-VLK-pNA, Molecular Innovation, Novi, MI) at 200 μM concentration in PBS was added, mixed, and immediately placed in a colorimetric plate reader (SpectraMax i3, Molecular Devices, San Jose, CA) set at 37° C. Absorbance at 405 nM was then monitored in each well over the next 5 minutes. Plasmin hydrolysis activity for each well was calculated from the fitted linear slope of the absorbance versus time curve over 5 minutes.
Preliminary studies confirmed concentration-dependent inhibition of plasmin activity by the antifibrinolytic aprotinin (Cat. #A1153, Sigma Aldrich, St Louis, MO) with an IC50 of approximately 500 nM (see
Plots of plasma standard volume versus log-transformed percent plasmin activity were consistently linear (R2>0.99) over the assay range (0 μL to 4 μL) (see
Stored DNA samples (N=2,298) from client-owned dogs were retrieved from the Washington State University Veterinary Teaching Hospital Patient DNA Bank and the Comparative Pharmacogenomics Laboratory Sighthound DNA Bank. DNA was extracted from buccal swab samples obtained by the hospital staff or by the dog's owner. Hospital patient samples were from dogs living in the Pacific Northwest of the United States, while the Sighthound DNA bank samples were obtained primarily by mail from dogs living throughout the United States. For hospital bank samples, the dog's breed was based on owner designation, whereas Sighthound Bank samples were verified by breed registration information. The 2,298 DNA samples represented 75 different dog breeds with a minimum of 10 DNA samples per breed and included 156 samples from mixed-breed dogs. The designation of a breed as belonging to the ‘Sighthound’ group was based on breed inclusion criteria for Sighthounds established by the American Kennel Club. Samples from greyhound dogs were divided into two breed subgroups based on whether they were identified by their owners as dogs bred for racing and registered with the National Greyhound Association (NGA; N=197) or were dogs bred for other purposes and registered with the American Kennel Club (AKC; N=64).
The instant example utilizes the methods described in Example 1 to identify delayed postoperative bleeding cases in canines and controls. Completed health surveys from the owners of 260 Scottish deerhounds were reviewed to identify delayed postoperative bleeding cases and controls. A flowchart illustrating the process used to select cases and controls is shown in
Case information for the seven dogs (SDH-1 to SDH-7) that met the inclusion/exclusion criteria for delayed postoperative bleeding are shown in Table 1.
Surgical procedures associated with delayed bleeding ranged from quite invasive procedures with the potential to cause extensive intraoperative bleeding, including splenectomy (SDH-2) and limb amputation (SDH-4), to moderately invasive procedures, including spays (SDH-1 and SDH-7), oophorectomy (SDH-5), and castration (SDH-3). One dog had experienced delayed bleeding after a relatively minor procedure involving removal of a sebaceous cyst from the skin (SDH-6). In addition to supportive care (primarily administration of intravenous fluids), most dogs (SDH-1 to SDH-6) with delayed postoperative bleeding were also treated with and responded positively to an antifibrinolytic drug. Specifically, bleeding was reported to have stopped within hours of drug administration. EACA (oral or injectable) was used in all instances. However, two dogs were not treated with any antifibrinolytic drug (SDH-1 and SDH-7). One of these dogs (SDH-1) recovered after 3 days of supportive care, while the other dog (SDH-7) died 2 days after a routine spay despite similar supportive care. Although a necropsy examination was performed on this latter dog (SDH-7), the report could not be obtained by the owner for review.
Complete and detailed veterinary medical records were available to review for two dogs (SDH-2 and SDH-3). SDH-2 was a 5-year-old male Scottish deerhound who underwent an exploratory laparotomy for symptoms including abdominal pain and radiographic evidence of splenic enlargement. Splenic torsion was diagnosed during surgery. A splenectomy was performed, as well as a prophylactic gastropexy. Recovery was uneventful until the morning after the procedure when both the hematocrit and the platelet count had significantly decreased (relative to immediate postoperative values) and continued to decrease throughout the day (see Table 2).
Frank blood was noted to be oozing from the abdominal incision. A moderate amount of fluid was also found in the abdominal cavity by ultrasound examination. EACA solution (750 mg) was diluted in Ringer's lactate solution and administered as an intravenous infusion. Both hematocrit and platelet counts significantly increased by the next morning (Day 2) and were within their normal range by Day 5. The dog recovered completely from the surgical procedure and was in good health at 8 years of age.
SDH-3 was a 1-year-old male Scottish deerhound diagnosed with unilateral cryptorchidism. Surgical castration was performed. Recovery was uncomplicated and the dog returned home the next day. However late on the second day after the procedure very heavy bleeding from the surgical site was observed by the owner. The dog was returned to the clinic where an exploratory surgery of the wound was performed. No single source of bleeding was identified, and the area was extensively cauterized. Following this surgery, in addition to intravenous fluids, antibiotics, and a single vitamin K injection, the dog was administered 500 mg of EACA orally, which was continued every 8 hours for 5 days. No further bleeding was observed by the owner and the dog recovered completely. Four years later, the same dog underwent an elective splenectomy surgery for suspected hemangiosarcoma. The dog received 500 mg of EACA as an intravenous infusion during the procedure, which was followed by a 500 mg oral dose every 8 hours for 5 days. No excessive bruising or delayed postoperative bleeding were observed and the dog completely recovered from the procedure. Examination of the spleen revealed multiple benign splenic hematomas rather than hemangiosarcoma. The dog was in good health at 8 years of age.
Two other dogs (SDH-4 and SDH-6) also had multiple surgical procedures with differing outcomes. SDH-4 was spayed at 1 year of age and had a surgical stabilization of a left front leg fracture at 6 years of age without evidence for delayed postoperative bleeding. However, at 6.5 years of age the dog's left foreleg was amputated after a diagnosis of osteosarcoma. The dog underwent the procedure without complication and was discharged the second day after surgery. Nevertheless, within 2 hours of returning home the owner noticed the appearance of severe bruising of the chest and abdomen with edema. The dog was readmitted where a low hematocrit value (26% compared to 61% before surgery) was found. The hematocrit continued to fall to 15% overnight. Based on a diagnosis of delayed postoperative bleeding, the dog was treated with a continuous intravenous infusion of EACA, as well as several transfusions of whole blood and plasma. The dog was discharged after 5 days with a hematocrit of 36%, which increased to 60% after one month. The dog had no further complications from the procedure. However, it was euthanized 6 months later because of paralysis associated with tumor metastasis to the spinal cord.
SDH-6 was surgically treated for a skin abscess resulting from a foreign body when 5 years old. The dog had received a pre-operative oral dose of EACA. No postoperative bleeding or bruising was observed. However, when 7 years old, a cutaneous sebaceous cyst was removed surgically without administration of prophylactic EACA. The next day after discharge, the owner observed excessive postoperative bleeding from the incision site. The dog was subsequently treated with orally administered EACA and the bleeding resolved. The dog recovered without further complication and was in good health at 9 years of age.
A further 55 Scottish deerhounds (SDH-C-1 to SDH-C-55) with available DNA samples were identified as controls for case-control genetic association analysis. The majority of dogs had undergone surgical desexing procedures (15 castrations, 13 spays, and 3 ovariectomies) or Caesarean section (n=15). Seven dogs were treated for major skin lacerations. The remaining 15 dogs underwent major abdominal or orthopaedic procedures, including cystotomies (n=4), gastric dilatation/volvulus surgery (n=2), splenectomy (n=1), fracture repair (n=4), anterior cruciate repair (n=1), patellar luxation correction (n=1), Achilles tendon repair (n=1), and a digit amputation (n=1). Thirteen dogs had multiple surgical procedures performed.
Gene-capture sequencing was used in an exploratory analysis to discover coding region variants in 7 candidate genes encoding proteins known to negatively regulate the fibrinolysis pathway to evaluate if mutations in one or more of these genes led to enhanced fibrinolysis (see
Ten single nucleotide variants (SNVs) were identified in 6 of the genes, while F13B contained no coding region variants (see
The genotypes for each of the SNVs in the discovery subset of cases and controls are shown in
SERPINF2 c.605 C>T was chosen for further study by case-control genotype association analysis since this variant showed the greatest effect on protein function and the strongest association with delayed bleeding phenotype in the discovery sample DNA subset.
The instant example utilizes the methods described in Examples 1 and 2 to demonstrate that SERPINF2 c.605 C>T genotype is associated with delayed postoperative bleeding. Briefly, the case-control genotype-phenotype association analysis was used to evaluate whether SERPINF2 c.605 C>T genotype was correlated with delayed postoperative bleeding phenotype. SERPINF2 c.605 C>T genotype results obtained by gene-capture sequencing were confirmed using a Taqman allele discrimination assay and extended to include all cases (n=7) and controls (n=55). As shown in Table 3, the variant SERPINF2 c.605 T allele frequency in cases was over 5 times that of control dogs (allelic OR=16; 95% CI=4.4 -57; P<0.0001).
None of the cases had the reference (SERPINF2 c.605 C/C) genotype, while none of the control dogs had the homozygous variant (SERPINF2 c.605 T/T) genotype. Compared to dogs with the reference SERPINF2 c.605 C/C genotype, the odds for postoperative bleeding were significantly higher for dogs with the heterozygous C/T genotype (genotypic OR=24; 95% CI=1.2-463; P=0.034) and with the homozygous variant T/T genotype (genotypic OR=567; 95% CI=9.7-33177; P=0.0023).
F7 c.407 G>A genotypes were also determined for the same case and control dogs (Table 4). In contrast to the SERPINF2 genotype results, there was no difference in the frequency of the variant F7 c.407 A allele in cases versus control dogs (allelic OR=0.98; 95% CI=0.2-−4.8; P=0.98).
The instant example utilizes the methods described in Examples 1 and 2 to demonstrate that SERPINF2 c.605 TT genotype is associated with lower plasma antiplasmin activities. Given the association of the SERPINF2 c.605 T allele with delayed postoperative bleeding, the function of alpha-2 antiplasmin (encoded by SERPINF2) was investigated to see if it would be decreased in the plasma of dogs with this variant. Since all commercial antiplasmin assays currently use human plasmin, a plasmin inhibition assay was developed using purified canine plasmin and validated with pooled Beagle dog plasma as a reference standard.
Retired racing greyhounds (greyhounds registered with the National Greyhound Association [NGA]) were studied because they have a relatively high SERPINF2 c.605 T allele prevalence and are reportedly predisposed to delayed postoperative bleeding. Out of 19 greyhounds genotyped, 3 dogs were SERPINF2 c.605 C/C, 10 dogs were SERPINF2 c.605 C/T and 6 dogs were SERPINF2 c.605 T/T. As shown in
The instant example demonstrates that reduced plasma antiplasmin activities in greyhounds with the SERPINF2 c.605 T/T genotype. This finding suggests that this variant (or perhaps another variant in close genetic linkage) negatively affects expression or function of the antiplasmin protein.
The instant example utilizes the methods described in Example 1 to demonstrate that the SERPINF2 c.605 T allele is primarily found in breeds susceptible to delayed postoperative bleeding. If the SERPINF2 c.605 C>T variant was causally linked to delayed postoperative bleeding, it would follow that the prevalence of this variant should be highest in those breeds known to be susceptible to this disorder, including retired racing greyhounds and Scottish deerhounds, as well as other sighthound dog breeds. Consequently, the prevalence and breed distribution of the variant SERPINF2 c.605 T allele was determined in 2,298 dogs representing 75 dog breeds (including 20 sighthound breeds) and 156 mixed-breed dogs. The SERPINF2 c.605 T allele frequencies are shown in
The SERPINF2 c.605 T allele was found in a higher proportion of sighthound dog breeds (15 of 20; 75%; see
see
The instant example utilizes genetic samples (e.g., DNA samples) obtained from animals for utilization according to the present disclosure. For instance, genetic samples can be collected from an animal via a swab or via blood according to general procedures known in the art. Thereafter, the genetic samples can be tested in a laboratory or other clinic.
For example, DNA can be extracted from the genetic sample and then tested via a PCR procedure. For instance, a Taqman SNP allele discrimination assay can be performed via PCR, including by real-time PCR. For this example, positive controls can be utilized comprising DNA samples from dogs with a known genotype (confirmed by Sanger sequencing). Further, negative controls can be utilized comprising samples with no DNA.
The following primers and probes can be utilized according to the instant example:
Following testing, genetic results of the animals can be reported as i) Normal/Normal (WT), ii) Normal/Mutant (HET), or iii) Mutant/Mutant (VAR).
Without being bound to any theory, it is believed that in Scottish deerhounds a dog identified as “VAR” has over a 500 times higher risk (compared with “WT” dogs) for developing delayed post-operative bleeding following surgery. Accordingly, it is believed that a prophylactic treatment with a plasmin inhibitor is beneficial to prevent post-operative bleeding in these dogs.
Without being bound to any theory, it is believed that in Scottish deerhounds a dog identified as “HET” dogs has a 25 times higher risk (compared with “WT” dogs) for developing delayed post-operative bleeding following surgery, especially if the dog is older in age. Consequently, it is believed that a prophylactic treatment with a plasmin inhibitor is beneficial to prevent post-operative bleeding in these dogs.
This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Ser. No. 63/152,219, filed on Feb. 22, 2021, the entire disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/017130 | 2/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63152219 | Feb 2021 | US |
