Causative alleles for chondrodysplastic dwarfism, factor XI deficiency, and factor XIII deficiency in the Kumamoto sub-breed of Japanese Brown cattle.

Japanese Brown cattle are the second most popular Wagyu breed, and the Kumamoto sub-breed shows better daily gain and carcass weight. One of the breeding objectives for this sub-breed is to reduce genetic defects. Chondrodysplastic dwarfism and factor VIII deficiency have been identified as genetic diseases in the Kumamoto sub-breed. Previously, we detected individuals in the Kumamoto sub-breed with causative alleles of genetic diseases identified in Japanese Black cattle. In the current study, 11 mutations responsible for genetic diseases in the Wagyu breeds were analyzed to evaluate the risk of genetic diseases in the Kumamoto sub-breed. Genotyping revealed the causative mutations of chondrodysplastic dwarfism, factor XI deficiency, and factor XIII deficiency and suggested the appearance of affected animals in this sub-breed. DNA testing for these diseases is needed to prevent economic loses in beef production using the Kumamoto sub-breed.

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Carrier frequency of autosomal recessive disorders (BC, BLAD, FXID and CVM) in Holstein cows in Jalisco, Mexico

The hereditary autosomal recessive disorders bovine citrullinemia (BC), bovine leukocyte adhesion deficiency (BLAD), factor XI deficiency (FXID), and complex vertebral malformation (CVM) have affected dairy cattle breeding significantly around the world. This study examined the carrier frequency of BC, BLAD, FXID, and CVM autosomal recessive disorders in Bos taurus Holstein cows bred in the Altos Norte region of the state of Jalisco, Mexico. We extracted DNA from 408 random samples of peripheral blood, and then used polymerase chain reaction (PCR) to identify insertion mutations for FXID, and PCR with restriction fragment length polymorphism (PCR-RFLP) for CVM, BC and BLAD. We visualized the PCR products using agarose gel electrophoresis stained with GelRed®. We found that 100% of wild-type (N/N) allele homozygous animals for genes CD18, ASS, and FXI were free of the mutations for BLAD, BC and FXID respectively. For gene SLC35A3 we estimated total carrier frequency of 10.3% and allele frequency of 5%.(AU)

Novel identification of Factor XI deficiency in Indian Sahiwal (Bos indicus) cattle.

Factor-XI deficiency (FXID) is inherited as autosomal lethal recessive disorder of carrier Holstein-Friesian bulls. A 76 base pair segment insertion into exon 12 in Factor-XI gene causes FXID in cattle. Keeping this in view the present study was conducted to screen breeding bulls of both indigenous and exotic breeds for mutation in Factor-XI gene and to find out the frequency of FXID carrier animals in breeding bulls. A total of 120 bulls of different age group maintained at Frozen Semen Bull Station, India were randomly selected from different cattle breeds to screen presence of FXID syndrome in breeding sires. Genomic DNA was isolated from blood of the selected bulls. PCR parameters were standardized to obtain 244 and 320 bp amplicons. The results showed that 2 Sahiwal bulls out of 120 animals were carrier for FXID. Amplicons of the carrier animals were sequenced and annoted, which confirms a 76 bp insertion in the exon 12. Bleeding and clotting time showed considerable discrepancy in the carrier animals as compared to the normal animals. The findings of relative mRNA expression of Factor XI transcript revealed identical tendency in the carrier. The frequency of carrier animals and mutant allele was 2.5 % and 0.025 respectively. This study recommends for screening of breeding at AI bull centers in the country for FXID. The study also stands a merit for identification of FXID carrier in Bos indicus for the first time.

A genetic predisposition for bovine neonatal pancytopenia is not due to mutations in coagulation factor XI.

Bovine neonatal pancytopenia (BNP) is a newly emerging disease in many European countries that causes haemorrhagic diathesis and mortality in neonatal calves. This study tested the hypothesis that genetic factors might be involved in BNP, since genetic defects resulting in coagulation disorders have been described in many species, including cattle. A familial pattern of occurrence of BNP cases was observed in an experimental population of cattle in Germany and BNP was diagnosed in nine calves on an experimental dairy herd from May 2007 to December 2009. All affected calves were descendents of a single F(1) sire in a specific F(2) resource population generated from Charolais and German Holstein bloodlines. Sequence analysis of the bovine coagulation factor XI (F11) gene as a functional candidate gene for BNP revealed an unusually high number of non-synonymous mutations within the gene compared to a whole genome mutation screen in cattle targetting random sequences. However, none of the mutations in the F11 gene were concordant with BNP status. Although these data and further pedigree analysis excluded a simple mode of inheritance of the BNP phenotype, there was a statistically significant (P=0.0001) accumulation of BNP cases in the specific pedigree examined, suggesting that a genetic component is involved in the development of BNP.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. METHODS: Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. RESULTS: Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). CONCLUSION: This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.

Identification of carriers of the mutation causing coagulation factor XI deficiency in Polish Holstein-Friesian cattle.

Factor XI (FXI) deficiency is a hereditary coagulation disorder observed in various mammalian species. The molecular basis of coagulopathy has been recognized in Holstein cattle as a 76-bp insertion in the coding region of the FXI gene. Because the disorder seems to have an impact on reproductive traits and udder health in cattle, we tested 103 randomly selected cows, 28 cows with repeat breeding, and 9 cows with recurrent mastitis for the presence of an abnormal FXI allele. Three related cows were diagnosed as carriers.

Identification of factor XI deficiency in Holstein cattle in Turkey.

BACKGROUND: Factor XI (FXI) is a plasma protein that participates in the formation of blood clots. Factor XI deficiency is autosomal recessive hereditary disorder that may be associated with excess bleeding in Holstein cattle. METHODS: In this study, 225 Holstein cows reared in Turkey were screened in order to identify FXI genotypes. DNA extractions were obtained from the fresh blood of the cows. Amplicons of FXI exon 12 were obtained by Polymerase Chain Reaction (PCR), and analyzed by 2% agarose gel electrophoresis stained with ethidium bromide. Additionally, all cows were confirmed by DNA sequencing to determine whether or not there was a mutant allele. RESULTS: Carriers of the FXI deficiency have two DNA fragments of 320 bp and 244 bp in size. The results of our study demonstrated that only four out of the 225 Holstein cows tested in Turkey carried the FXI deficiency. The frequency of the mutant FXI allele and the prevalence of heterozygous cows were found as 0.9% and 1.8%, respectively. CONCLUSION: The DNA-based test determines all genotypes, regardless of phenotype or FXI activity. The mutation responsible for the FXI deficiency had not been detected in Holstein cattle in Turkey before prior to this study. The frequency of the mutant FXI allele needs to be confirmed by carrying out further analyses on cattle in Turkey and the selection programs should be developed to eliminate this genetic disorder.

Genetic description of factor XI deficiency in holstein semen in Western Japan.

Factor XI deficiency was detected in Holstein cows and mummified foetuses in Japan; however, no report is available about the occurrence of Factor XI deficiency in Holstein semen in Japan. Five hundred cows in twelve dairy farms in Hiroshima Prefecture, Japan were under the study. Genomic DNA was extracted from the cows using a commercial DNA kits and screened to Factor XI mutation. Based on the information of the carrier cows found in the cattle population, four Holstein bulls were analysed for Factor XI mutation. DNA was extracted from bull's semen using phenol chloroform method. Extracted genomic DNA of the bull's semen was typed for Factor XI using specific polymerase chain reaction (PCR) primers. The resultant PCR was sequenced using big dye terminator sequencing method. The pedigree of the bulls was investigated. Furthermore, the inheritance of Factor XI mutation to next generation was estimated. Out of the 500 cows, five were heterozygous to Factor XI. Moreover, out of the four bulls, one was found to carry the mutation of Factor XI; it was also a complex vertebral malformation (CVM) carrier. In DNA sequencing, the insertion mutation of 76 bp of poly-adenine that characterizes the Factor XI deficiency was detected in the carrier bull as well as the carrier cows. Pedigree analysis of the carrier bull revealed that his father and mother ID were 2247419A and 14189172A, respectively, that originated from USA Holstein. Out of six daughter cows born to the carrier bull, one cow (16.6%) inherited Factor XI mutation, while three of them (50.0%) inherited CVM mutation. Autosomal recessive genes that affect cow's reproduction have a particular concern to dairy industry. To our knowledge this is the first report of Factor XI mutation in Holstein semen in Japan.

Pedigree analysis of factor XI deficiency in Japanese black cattle.

Using a DNA-based diagnostic test for factor XI deficiency in Japanese black cattle, we surveyed 123 cattle (42 sires and 81 dams) in Gifu and Hyogo prefectures, and calculated gene frequencies. In sires, we drew up the pedigree network of the cattle with the factor XI deficiency. Results showed that the mutated allele of factor XI deficiency was retroactive in at least 6 or more generations of sires. Frequencies of the mutant gene were higher at 26.4% in total, and at 33.3% in sires. All 7 cattle with the homozygote of mutated allele were clinically normal, and showed no bleeding episodes. The mutated allele of factor XI deficiency might be widespread among Japanese black cattle.

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency.

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

Carrier rate of Factor XI deficiency in stunted Japanese black cattle.

Blood examinations and genotyping of Factor XI (F11) were performed in growth retardation Japanese Black cattle and their dams. Genotyping of F11 revealed that the recessive homozygous and heterozygous genotype frequencies were 5.2% and 50.0% in the Claudin-16 (CL-16) deficiency group (n=58), 0% and 14.2% in the renal dysplasia group (n=7), 0% and 26.1% in the non-CL-16 deficiency nephritis group (n=23), 8.9% and 46.7% in the hypogenesis syndrome group (n=45), 6.2% and 25.0% in the neonatal weak calf syndrome group (n=32), 9.1% and 38.6% in the respective dams group (n=44), 0% and 23.1% in the normal cattle group (n=13), and 5.9% and 38.2% in total (n=222), respectively. These results showed that the carrier rate of F11 deficiency was high in Japanese Black cattle, and that the CL-16 deficiency, hypogenesis syndrome, neonatal weak calf syndrome, and dams groups had a large amount of recessive homozygous genotype than the other groups. No abnormal bleeding was observed clinically in the present study, and 4 of the recessive homozygous dams showed normal growth and parturition.

Simulation of normal, carrier and affected controls for large-scale genotyping of cattle for factor XI deficiency

An insertion mutation within exon 12 of the factor XI gene has been described in Holstein cattle. This has opened the prospect for large-scale screening of cattle using the polymerase chain reaction (PCR) technique for the rapid identification of heterozygous animals. To facilitate such a screening process, the mutant and normal alleles of factor XI gene, represented by 244- and 320-bp PCR amplified fragments, were individually cloned in Escherichia coli using a multicopy plasmid cloning vehicle to generate pFXI-N and pFXI-M, respectively. The authenticity of the inserts was confirmed by nucleotide sequencing. A nested PCR method was developed, by which PCR amplicons generated from primers with annealing sites on the recombinant plasmids and by flanking the insert were used as templates for amplification of the diagnostic products using factor XI gene-specific primers. An equimolar mixture of both PCR amplicons, originating from pFXI-N and pFXI-M, constituted the carrier control while the individual amplicons were the affected and normal controls. The controls were used as references for in-gel comparison to screen a population of 307 cattle and 259 water buffaloes; the frequency of the mutant allele was found to be 0. No DNA size standards were required in this study. The simulated control DNA samples representing normal, carrier and affected cattle have the potential to help in large-scale screening of a cattle population for individuals that are carriers or affected by factor XI deficiency.

Factor XI mutation in a Holstein cow with repeat breeding in Japan.

Factor XI deficiency is an autosomal recessive coagulopathy in Holstein cattle. Affected cows have a tendency to show repeat breeding. Forty repeat breeding Holstein Friesian cows were selected and tested for the Factor XI mutation. Genomic DNA was isolated from the blood of the cows (n=40). Exon 12 of the Factor XI gene of the cows was amplified by PCR. One repeat breeding cow was heterozygous to the Factor XI mutation as indicated by the presence of two DNA fragments of 320 bp and 244 bp. The insertion of the 76 bp in the heterozygous cow was confirmed by DNA sequencing. The heterozygous cow was in her fourth lactation. She gave birth to male twins at the last calving. She was inseminated artificially four times after the last calving. Factor XI deficiency in cattle has been reported in different countries. However, no case was reported in Japan. This might be the first to report Factor XI mutation in Holstein cattle in Japan.

An insertion mutation of the bovine Fii gene is responsible for factor XI deficiency in Japanese black cattle.

Factor XI deficiency in Japanese black cattle is an hereditary mild bleeding disorder with an autosomal recessive mode of inheritance. To characterize the molecular lesion causing factor XI deficiency in cattle, we isolated an entire coding region of the bovine F11 gene, which comprises 15 exons and 14 introns, and determined its nucleotide sequences. Comparison of the nucleotide sequences of the F11 gene between affected and unaffected animals revealed an insertion of 15 nucleotides in exon 9 of the affected animals. The insertion results in a substitution of one amino acid with six amino acids in a highly conserved amino acid sequence in the fourth apple domain of factor XI protein. Genotyping of the F11 gene in 109 Japanese black cattle revealed that the insertion clearly corresponded to the factor XI activities of the animals. We therefore concluded that the insertion of 15 nucleotides in the F11 gene is the causative mutation for factor XI deficiency in Japanese black cattle. Genotyping of the F11gene by detecting the insertion will be an effective DNA-based diagnostic system to prevent incidence of the disease.

DNA extraction from bovine mummified fetuses and detection of factor XI gene deficiency in the mummies.

Genomic DNA extracted from bovine mummified tissue is valuable material for detection of some genes that may contribute to fetal abnormalities. In this study bovine genomic DNA was extracted from the hardened tissue samples of ten bovine mummified fetuses. The amount of genomic DNA extracted from 2 g of the mummified tissues by the phenol/chloroform-ethanol method was low (less than 4 microg/ml) for all samples. The extracted DNA was then amplified by the GenomiPhi DNA amplification system. After amplification, the amount of DNA was increased to more than 100 microg/ml for all samples. This amplification system was shown to be a good tool for amplifying the genomic DNA of the mummified fetuses. The amplified genomic DNA was used for testing the mummies for Factor XI gene deficiency, an autosomal recessive deficiency involved in the early stages of the intrinsic blood coagulation pathway. Exon 12 of the Factor XI gene of the mummies was amplified by PCR. Two of the ten mummified fetuses were heterozygous for the Factor XI gene as indicated by the presence of two amplified DNA fragments of 320 bp and 244 bp. Factor XI deficiency has already been described in Holstein cattle. However, no report is available for bovine fetus. In this study, DNA was extracted and amplified from the bovine mummified fetuses, and the samples were successfully tested for Factor XI gene deficiency in the mummies.

Identification of a mutation associated with factor XI deficiency in Holstein cattle.

An autosomal recessive deficiency of blood coagulation factor XI (FXI) has been described in Holstein cattle. Current testing methods are unsuitable for accurately identifying carriers (heterozygotes) of the disease. To identify the molecular basis of this deficiency, a polymerase chain reaction (PCR)-based strategy was implemented to clone and sequence the bovine FXI gene (F11) from animals of different genotypes. Approximately 14 kb of genomic DNA sequence and 1.8 kb of cDNA sequence, corresponding to exon 3 through the 3'-UTR, of the bovine gene were obtained. Comparison of sequences derived from homozygous normal and deficient individuals revealed that FXI deficiency in Holsteins is associated with the insertion of a 76 bp segment [AT(A)(28)TAAAG(A)(26)GGAAATAATAATTCA] within exon 12. This insertion introduces a stop codon that results in a mature FXI protein lacking the functional protease domain encoded by exons 13, 14 and 15. Based on these data, a DNA-based diagnostic test has been developed for accurate genotyping. Using this method, the frequency of the mutated allele has been determined to be 1.2% in a contemporary population of the USA Holstein sires.

Congenital factor XI deficiency in a domestic shorthair cat.

A 6-month-old, female, domestic shorthair cat was examined after onychectomy and ovariohysterectomy because of bleeding from the paws. Prolonged activated partial thromboplastin time was discovered, Coagulation factor analyses revealed deficiency of factor XI coagulant activity. Plasma mixing studies indicated factor deficiency or dysfunction rather than factor inhibition. Feline factor XI deficiency in one adult cat has been previously reported but was attributed to factor XI inhibitors. The signalment, lack of primary disease, and the finding of persistent factor XI deficiency in the absence of coagulation inhibitors were considered compatible with congenital factor XI deficiency in the cat of this report.

Comparison of in vitro function of neutrophils from cattle deficient in plasma factor XI activity and from normal animals.

Cattle, homozygous for the genetic disorder of factor XI (FXI) deficiency, exhibit less than 2% of normal plasma FXI activity, display an increased bleeding tendency and are more prone to infectious diseases. FXI is one of the protein components of the contact activation system of coagulation that assembles on the surface of circulating neutrophils. Because of the central role of neutrophils in inflammation, the in vitro responses of neutrophils from normal and FXI deficient cattle were compared. Neutrophil degranulation was evaluated by measuring the release of myeloperoxidase and alkaline phosphatase, and the respiratory burst was evaluated by determining superoxide anion production. Neutrophils from FXI deficient animals exhibited a significant increase (P < 0.05) in the spontaneous release of granule contents compared to the cells from normal cattle. Following stimulation with C5a complement derived from normal serum, the neutrophils from the FXI deficient animals exhibited a greater increase (P < 0.05) in both alkaline phosphatase release and superoxide production. In these neutrophils, following stimulation with C3b complement from normal serum, the relative increase in myeloperoxidase release compared to the unstimulated neutrophils was lower than that observed in the neutrophils from normal animals. There was minimal superoxide production in unactivated neutrophils from either normal or FXI deficient cattle and the response to phorbol ester stimulation was similar in both groups of animals. The C5a complement from FXI deficient serum was more effective (P < 0.05) in stimulating alkaline phosphatase release and superoxide production in normal neutrophils than the equivalent fraction from FXI deficient serum while the C3b complement from the FXI deficient serum was less effective than the normal serum fraction at inducing myeloperoxidase release from normal neutrophils. The results indicate that the differences in the in vitro neutrophil function are likely related to a variation in the function of the contact activation system on the neutrophil surface between normal and FXI deficient animals.

[Applicability of activated partial thromboplastin time (APTT) as a screening test of milk to medium deficiencies of coagulation factors in cats]. / Eignung der aktivierten partiellen Thromboplastinzeit (aPTT) als Screeningtest für gering- bis mittelgradige Gerinnungsfaktorverminderungen bei der Katze.

The present investigation examined if the aPTT shows sufficient sensitivity for single factor activities also in cats when measured with the test optimized for humans using a commercial reagent. Comparative measurements were done with different modifications of the aPTT (sample predilution, addition of fibrinogen). Measurements of the aPTT using different methods and the activity of the coagulation factors II, V, X, VIII, IX, XI, and XII were performed in 42 healthy cats in order to determine the reference ranges. The same measurements were done on 21 cat plasmas where at least one of the coagulation factors was diminished in relation to the corresponding reference range. The conventional aPTT reflected the decrease in coagulation factor activity in each of the 21 plasmas by prolongation above the reference range (14.6-24.4 s). The test thus possesses high sensitivity and is a suitable screening test also for the cat. In contrast, in tests with sample predilution the sensitivity was lower (3-4 false negative results). This was probably caused by the distinct increase of the range of the reference values.