John A. Gerlach

Towards construction of a canine linkage map: Establishment of 16 linkage groups

1Norwegian Kennel Klub and Department of Morphology, Genetics and Aquatic Biology, Section of Genetics, P.O. Box. 8146 Dep., N-0033 Oslo, Norway ZDepartment of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7023, 750 07 Uppsala, Sweden 3Department of Animal Science and Animal Health, Division of Animal Genetics, The Royal Veterinary and Agricultural University, Btilowsvej, 13, DK-1870, Fredriksberg C, Copenhagen, Denmark ~The Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK 5Centre for Preventive Medicine, Animal Health Trust, PO Box 5, Newmarket, Suffolk CB8 7DW, UK 6Medical Technology and Medicine, Michigan State University, B228 Life Science, East Lansing, Michigan 48824-1317, USA 7Institute of Animal Breeding, University of Berne, Bremgartenstrasse 109 a, 3012 Berne, Switzerland

Possible pathogenic pathways in the adverse clinical events seen following ivermectin administration to onchocerciasis patients.

Background Reactions are commonly associated with the chemotherapy of onchocerciasis. However unmanageable reactions are uncommon when ivermectin (Mectizan®) is used for the treatment of this infection, and this drug has proved to be a great improvement over previously used agents. Serious adverse events (SAE) nevertheless have occurred, and there is considerable concern about the negative effect such events may have on mass drug administration programs. This paper reviews the basic pathogenic mechanisms that can be involved in the destruction of microfilaria by chemotherapeutic agents. A central challenge to filarial chemotherapy is the need to remove parasites from biologically sensitive tissues, a more difficult medical challenge than eliminating nematodes from the gastrointestinal tract. Explanations for the etiology of the serious adverse reactions occurring with ivermectin treatment in specific geographic areas where there is coincident heavy Loa loa infections are hampered by a lack of specific pathological case material. Ways to investigate these possibilities are reviewed. Possible pathogenic mechanisms include embolic vascular pathology accompanied by local inflammation, blood brain barrier mdr1 abnormalities, and genetic predisposition to excessive inflammatory responses. Conclusion It is important to keep ivermectin, and all its associated adverse clinical events, in perspective with the many other chemotherapeutic agents in general use – many of which produce serious adverse events even more frequently than does ivermectin. Currently available evidence indicates that the pathogenesis of the Loa-associated adverse reactions are probably related to inflammatory responses to microfilariae in specific tissues. However, the possibility of genetic predispositions to pathology should also be considered.

HLA‐E*0103X is associated with susceptibility to Pemphigus vulgaris

Non‐classical human leucocyte antigen‐E (HLA‐E) mediates natural killer and CD8+ T‐cell activity, suggesting a role in the regulation of autoimmunity. HLA‐E*0103X/*0103X has been associated with Behcets disease and HLA‐E *0101/*0103X with childhood onset diabetes. We investigated HLA‐E allele status in 52 Caucasian and Ashkenazi Jewish Pemphigus vulgaris (PV) patients and 51 healthy controls by restriction fragment length polymorphism–polymerase chain reaction and amplification refractory mutation system. Associations were determined via chi‐square test, Fishers exact test and logistical regression analysis. HLA‐E outcomes included presumed homozygous *0101/*0101 or *0103X/*0103X genotype status or *0101/*0103X heterozygous status. PV did not significantly associate with either *0101/*0101 or *0101/*0103X genotypes. HLA‐E*0103X/*0103X (presumed homozygote) is significantly increased in patients with PV versus controls (P = 0.0146, OR = 3.730, 95%CI = 1.241–11.213). Our data provide the first evidence that HLA‐E*0103X is a marker for genetic risk in PV.

Increased representation of the PTPN22 mutation in patients with immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by antibody-mediated platelet destruction. Why patients produce an antibody against their own platelets is poorly understood. Twin and family studies suggest that some patients may have a genetic predisposition towards development of ITP [1,2]. A protein tyrosine phosphatase (ptpn22) present in lymphocytes is an important negative regulator of signal transduction for the T-cell receptor–MHC complex and has been associated with autoimmune disorders that produce autoantibodies [3–7]. As ITP patients produce antibodies against their own platelets, we hypothesize that the frequency of the mutation 1858C>T in the PTPN22 gene observed in ITP patients would be increased like the findings reported for other immune disorders. The investigative protocol was approved by the institutional review board of Michigan State University and all patients signed informed consent for genetic testing. Blood samples for genotyping were obtained from 45 unrelated consecutive patients diagnosed with ITP according to the recent definition of the international working group, and who had a positive quantitative direct antiplatelet antibody of at least 500 molecules IgG per platelet [8]. Additional inclusion criteria were: a platelet count below 100 000, a marrow morphology, when obtained, compatible with the diagnosis of ITP, no concomitant autoimmune disorders, no medications known to cause thrombocytopenia, no associated viral disorders and no current diagnosis of malignancy. The frequencies of the PTPN22 mutation observed in a population of 926 normal subjects from New York City, (Table 1) as well as the frequencies reported for the immunological disorders listed in (Table 2), were used for statistical comparisons. Genotyping of PTPN22 1858C/T SNP was performed by PCR-restriction fragment length polymorphism (RFLP) [9]. A radiometric technique was used for quantitation of autologous platelet surface IgG [10]. Demographic data describing the patients age and race or ethnic background were obtained from the patients. The allele and genotype frequencies among patients with ITP, historical controls and the other immune disorders referenced in Tables 1 and 2 were compared using the v-test. A P -value < 0.05 was considered statistically significant. Of the 45 ITP patients studied, 26 (58%) were male and 19 (42%) were female. Median age was 43 years (range, 29–89). Race or ethnic background, as reported by the patients, was 39 Caucasian and 6 Hispanic. Twelve ITP patients (27%) were Table 2 PTPN22 percent positive in ITP patients compared with other immune disorders

6 - Nomenclature for Factors of the Dog Major Histocompatibility System (DLA), 2000

This chapter provides nomenclature for factors of the dog histocompatibility system (DLA). The Major Histocompatibility Complex (MHC) of the dog and other Canidae appear to be highly polymorphic; and alleles of these genes are likely to be functionally relevant in regulating the immune response and the susceptibility/resistance to immune-mediated diseases. Since the first nomenclature report, 48 DLA-88 , 16 DLADRB1 , 6 DLA-DQA1 , and 15 DLA-DQB1 alleles have been named. Although there is some evidence for 2, 3, and 4 alleles respectively for DLA-12 , DLA-64 , and DLA-79 , no sequence alignments have been published, and no alleles have been lodged in GenBank till date. The principles for naming the MHC genes and alleles in different Canidae, and its application to sub-species and hybrids, have been considered. The principles established here for the Canidae group may have applications in other animals that have been domesticated. Compelling evidence of MHC identity exists among these Canidae, because three locus haplotypes are shared among dogs, wolves, and coyotes.

PTPN22 profile indicates a novel risk group in Alopecia areata.

Alopecia areata (AA) is a genetically determined autoimmune hair loss disorder. A polymorphism in protein tyrosine phosphatase N22 (PTPN22), which normally suppresses T-cell proliferation, has been associated with human autoimmune disease, including AA in European populations. PTPN22 genotype frequency in known to vary geographically. Accordingly, we conducted a case-control study of the PTPN22 1858C/1858T (C1858T) genotype frequency in North American Caucasians and non-Caucasians. Allele status was determined in 365 AA patients, 196 healthy related control subjects (RC) and 77 unrelated healthy control subjects (UrC). We found that AA patients are more likely to carry the PTPN22 C1858T genotype than UrCs (p = 0.075), and this association reached significance in patients with the most severe disease presentation (Alopecia universalis vs. UrC, p = 0.024). PTPN22 C1858T genotype frequency in RC did not differ from AA patients (p = 0.657), but was significantly increased in comparison with UrC (p = 0.050). PTPN22 1858C/T genotype frequency increased in related control subjects most closely associated with patients (one family members of AA patients vs. UrC subjects, p = 0.040). Our data suggests that AA patients (particularly those that are severely affected) and closely related control subjects may belong to a shared inheritance group with increased disease risk, distinct from secondary and tertiary relatives and unrelated individuals. These findings have implications for the study of candidate genes and susceptibility to AA that may influence future clinical monitoring of unaffected, but closely related family members of patients.

PTPN22 1858T is not a risk factor for North American pemphigus vulgaris.

Abstract:  Alterations in the protein tyrosine phosphatase N22 (PTPN22) gene affect the threshold for lymphocyte activation. The PTPN22 1858T polymorphism leads to uninhibited T‐cell receptor cascade propagation. An elevated PTPN22 1858C/T genotype frequency has been correlated with several autoimmune disorders which have T‐cell and humoral components. However, a recent Tunisian report demonstrated no association between PTPN22 1858T and patients with Pemphigus vulgaris (PV), an autoantibody‐associated blistering disorder. Because PTPN22 1858T allele frequency is known to vary across ethnic populations, we conducted a case–control study investigating the relationship between PTPN22 1858T and PV in North American patients of either Ashkenazi Jewish or Caucasian (non‐Ashkenazi) decent. Participant genotype was determined in 102 PV patients and 102 healthy controls by restriction fragment length polymorphism–polymerase chain reaction genotyping. Relationships were calculated using Fisher’s exact tests and chi‐squared tests. We report that the PTPN22 1858C/T genotype is not significantly associated with PV in either Caucasians (P = 0.83) or Ashkenazi Jews (P = 0.60). Further stratification of the patient population by gender, age of disease onset, HLA‐type, family history of autoimmune disease, history of anti‐desmoglein (anti‐Dsg) 3 or anti‐Dsg1 antibody response, history of lesion morphology, and disease duration did not uncover significant associations between the PTPN22 1858T allele and PV subgroups. Our data indicate that the PTPN22 1858T mutation is not associated with PV in the North American population. We do observe an elevation of PTPN22 1858C/T genotype frequency in male PV patients. Further investigation will be required to determine if this trend reaches significance in larger studies.

Anti-thyroid peroxidase reactivity is heightened in pemphigus vulgaris and is driven by human leukocyte antigen status and the absence of desmoglein reactivity

Pemphigus vulgaris (PV) belongs to an autoimmune disease cluster that includes autoimmune thyroid disease (AITD), suggesting common mechanisms driving autoimmune susceptibility. Our group has shown that PV patients exhibit significant reactivity to AITD-related anti-thyroid peroxidase (anti-TPO), and anti-TPO antibodies affect signaling pathways in keratinocytes similar to anti-desmoglein (Dsg) 3 antibodies. To further assess the relevance of anti-TPO reactivity in PV, we analyzed anti-TPO levels in 280 PV and 167 healthy control serum samples across a comprehensive set of variable and static parameters of disease activity and etiopathogenesis. PV patients have significantly higher activity rates (A.R.s) for anti-TPO than healthy controls, but levels do not differ between phases of clinical activity and remission. Patients that carry both the PV-associated human leukocyte antigen (HLA) alleles DRB1*0402 and DQB1*0503, or DQB1*0503 alone show a low prevalence of anti-TPO (A.R. 9.5 and 4.8%, respectively), while patients that lack expression of these alleles or carry DRB1*0402 alone have a much higher prevalence of anti-TPO (A.R. 23.1 and 15.8%, respectively), suggesting that the absence of DQB1*0503 may predispose patients to the development of anti-TPO antibodies. Similarly, anti-Dsg1−/3− patients have a higher anti-TPO A.R. (26.9%) than anti-Dsg1−/3+ (18.8%), anti-Dsg1+/3− (14.3%), and anti-Dsg1+/3+ (3.9%) patients. Our data suggest that anti-TPO reactivity in PV is driven by genetic markers that may be in linkage disequilibrium with the established PV-susceptibility alleles and that this association drives the selection of a combination of anti-Dsg and anti-TPO antibodies, with anti-TPO filling the gap in active patients that do not carry the established PV-associated autoantibodies and/or are lacking the established PV-HLA-susceptibility alleles.

Further characterization of HLA homozygous typing cell lines at the LMP2 polymorphic codon 60 by an ARMS typing method.

LMP2 is a subunit of the 20S proteasome within the cellular cytosolic compartment that is thought to cleave proteins into approximately 9 amino acid long oligopeptides. It is hypothesized that changes in the low molecular mass protease (LMP) gene sequence may alter the activity or specificity in which the LMP genes cleave peptides. Currently, the typing method for LMP2 involves polymerase chain reaction (PCR), restriction enzyme digestion, and gel electrophoresis. To help reduce the cost and cumbersomeness of this method, a new typing method was adapted for the LMP2 gene. To establish this new amplification refractory mutation system (ARMS) typing method, primers have been defined, amplification conditions optimized, and control cell lines sequenced to validate testing parameters. Results are listed for selected 10th and 11th International Histocompatibility Workshop homozygous cell lines.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

BACKGROUND In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). OBJECTIVE The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. METHODS Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. RESULTS Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. CONCLUSIONS Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes.