Imprinting of canine IGF2 and H19.

Genomic imprinting occurs in therian mammals and is a phenomenon whereby the two alleles of a gene are differentially expressed, based on the sex of the parent from whom the alleles were inherited. The allelic differences in expression are the consequence of different epigenetic modifications that are established in the sperm or oocyte during gametogenesis and transmitted at fertilization to offspring. A small minority of genes is regulated in this way but they have important biological functions, and aberrant regulation of imprinted genes contributes to disease aetiology in humans and other animals. The factors driving the evolution of imprinted genes are also of considerable interest, as these genes appear to forego the benefits of diploidy. To broaden the phylogenetic analysis of genomic imprinting, we began a study of imprinted genes in the domestic dog, Canis familiaris. In this report, we show that canine IGF2 and H19 are imprinted, with parent-of origin-dependent monoallelic expression patterns in neonatal umbilical cord. We identify a putative imprint control region associated with the genes, and provide evidence for differential methylation of this region in a somatic tissue (umbilical cord) and for its hypermethylation in the male germline. Canis familiaris is fast becoming a highly informative system for elucidating disease processes and evolution, and the study of imprinted genes in this species may help in understanding how these genes contribute to the generation of morphological and behavioral diversity.

Qualitative and semiquantitative assessment of thyroid hormone binding proteins in greyhounds and other dog breeds.

Total thyroxine (T4) concentrations are lower in healthy greyhounds compared to most other non-sighthound breeds. In humans, variations in the structure or concentration of the major thyroid hormone binding proteins are responsible for most reported differences between total T4 concentrations in healthy individuals from different ethnic groups or other subpopulations. The aim of this study was to determine if such variations are also responsible for the lower total T4 concentrations in greyhounds. The predicted protein sequences of thyroxine-binding globulin (TBG), transthyretin and albumin were determined in liver tissue from a euthyroid greyhound with decreased T4 concentration and a Jack Russell terrier using reverse-transcriptase PCR. Sequences were compared to each other and online reference sequences. Serum proteins from 21 greyhounds and 21 non-sighthound dogs were separated by denaturing electrophoresis and immunoblots probed with polyclonal antibodies to human TBG and transthyretin. Reactive bands were quantified by densitrometry, expressed relative to the mean of reference samples included in each gel. Serum albumin concentrations were measured using a commercially-available assay. Several SNPs were identified but none was thought likely to explain the lower total T4 concentrations in greyhounds. There was no significant difference between the quantity of any of the binding proteins in serum from greyhounds and non-sighthound dogs. However, total T4 and transthyretin concentrations were highly correlated in the greyhound group (r = 0.73, P = 0.0002). Variation in the sequence of thyroid hormone binding proteins is not responsible for low greyhound total T4 concentrations. Further evaluation of the role of transthyretin is warranted.

Canine MAS1: monoallelic expression is suggestive of an imprinted gene.

Imprinted genes are epigenetically modified in a parent-of-origin dependent manner and as a consequence are differentially expressed, with one allele typically expressed while the other is repressed. In canine, the insulin like growth factor 2 receptor gene (IGF2R) is imprinted with predominant expression of the maternally inherited allele. Because imprinted genes usually occur in clusters, we examined the allelic expression pattern of the gene encoding the canine Mas receptor (MAS1), which is located upstream of IGF2R on canine chromosome 1 and is highly conserved in mammals. In this report we describe monoallelic expression of canine MAS1 in the neonatal umbilical cord of several individuals and we identify the expressed allele as maternally inherited. These data suggest that canine MAS1 is an imprinted gene.

Major histocompatibility complex class II alleles and haplotypes associated with non-suppurative meningoencephalitis in greyhounds.

Non-suppurative meningoencephalitis is a breed-restricted canine neuroinflammatory disorder affecting young greyhounds in Ireland. A genetic risk factor is suspected because of the development of disease in multiple siblings and an inability to identify a causative infectious agent. The aim of this study was to examine potential associations between dog leucocyte antigen (DLA) class II haplotype and the presence of the disease. DLA three locus haplotypes were determined in 31 dogs with non-suppurative meningoencephalitis and in 115 healthy control dogs using sequence-based typing (SBT) methods. All dogs were unrelated at the parental level. Two haplotypes (DRB1*01802/DQA1*00101/DQB1*00802 and DRB1*01501/DQA1*00601/DQB1*02201) were significantly (P = 0.0099 and 0.037) associated with the presence of meningoencephalitis, with odds ratios (95% confidence interval) of 5.531 (1.168-26.19) and 3.736 (1.446-9.652), respectively. These results confirm that there is an association between DLA class II haplotype and greyhound meningoencephalitis, suggesting an immunogenetic risk factor for the development of the disease. Greyhound meningoencephalitis may be a suitable model for human neuroinflammatory diseases with an immunogenetic component.

Clinical and clinicopathological features of non-suppurative meningoencephalitis in young greyhounds in Ireland.

The clinical and clinicopathological features of non-suppurative meningoencephalitis in 30 greyhounds were reviewed. The dogs were from 21 separate litters, comprised both sexes (16 males and 14 females) and ranged in age from five to 18 months. In 14 (66.7 per cent) litters, more than one case was suspected or confirmed, and the number of siblings affected within individual litters ranged from one to seven. Clinical signs were progressive and varied from five days to 12 months in duration; 12 dogs had signs of two weeks' duration or less. The rate of progression of signs was variable. Common features included dullness or lethargy (22), altered behaviour (21), proprioceptive and postural reaction deficits (18), circling (17), ataxia (17), decreased appetite (15) and weight loss (13). No consistent haematological or biochemical abnormalities were identified and serology failed to implicate Toxoplasma gondii or Neospora caninum. Cerebrospinal fluid analysis revealed mild or moderate mononuclear pleocytosis in 12 (70.6 per cent) of 17 dogs. No definitive antemortem diagnosis could be made in any affected dog.

Epigenetics and gene expression.

Transcription, translation and subsequent protein modification represent the transfer of genetic information from the archival copy of DNA to the short-lived messenger RNA, usually with subsequent production of protein. Although all cells in an organism contain essentially the same DNA, cell types and functions differ because of qualitative and quantitative differences in their gene expression. Thus, control of gene expression is at the heart of differentiation and development. Epigenetic processes, including DNA methylation, histone modification and various RNA-mediated processes, are thought to influence gene expression chiefly at the level of transcription; however, other steps in the process (for example, translation) may also be regulated epigenetically. The following paper will outline the role epigenetics is believed to have in influencing gene expression.

Genomic imprinting in Canis familiaris.

For the vast majority of mammalian genes, maternally- and paternally-derived alleles behave identically and are either expressed or repressed, regardless of whether they were inherited from egg or sperm. For imprinted genes, however, this is not the case. The alleles of imprinted genes are epigenetically modified in a parent-of-origin-specific manner and, as a consequence, maternally- and paternally-derived alleles behave differently. Typically one allele is expressed while the other is silent. Although relatively few in number, imprinted genes are the focus of intensive study, as they have important roles in embryonic development. Abnormal expression of imprinted genes results in growth disorders and is implicated in several clinical conditions. Most studies of imprinted genes have been performed in rodents or primates, with limited studies in other mammals such as bovine and opossum. We have recently demonstrated the existence of imprinted genes in the canine, by showing that the canine insulin-like growth factor 2 receptor gene (IGF2R) is monoallelically expressed, with predominant expression of the maternally-derived allele and repression of the paternally-inherited allele. Our ultimate goal is to characterize all imprinted genes in the canine, and to understand how they contribute to canine reproduction, development and disease. Such knowledge will be vital for optimizing the success of most reproductive strategies in the canine.

Cultural case management of latent tuberculosis infection.

SETTING: The proportion of tuberculosis (TB) among foreign-born individuals in the United States is steadily increasing. Treatment of latent TB infection can prevent future cases of disease, although generally only 60% of patients who start a 6-month regimen of isoniazid complete therapy. OBJECTIVE: Cultural case management--employing case manager cultural mediators who serve patient-defined needs in addition to performing TB control functions--may improve results of testing and treatment in one high-risk group, new refugees. DESIGN: A cultural case management approach was established for finding and treating latent TB infection among three groups of new refugees: from the former Soviet Union (FSU), former Yugoslavia (FY), and Somalia. RESULTS: From July 1999 through December 2000, treatment was offered to 442 refugees, of whom 389 (88%) started and 319 (82%) completed therapy. The completion rate among starters from the FSU was 76%, for those from FY it was 94% and for those from Somalia it was 88%. Among all refugees to whom treatment was offered, 319/442 (72%) completed therapy. CONCLUSION: Cultural case management may be a useful tool for expanding treatment of latent TB infection among foreign-born individuals.

Analysis of the frequency distribution of tuberculin skin test readings: a tool for the assessment of group contact investigations.

SETTING: The public health tuberculosis control program covering Seattle, Washington, and its surrounding suburban areas. OBJECTIVE: To describe a tool of potential usefulness in the assessment of transmission of tuberculosis in contact investigations of groups, such as co-workers or schoolmates of an infectious case, with a low prior probability of latent tuberculosis infection (LTBI). DESIGN: Tuberculin skin test (TST) readings in mm of the group being tested were graphed and compared with the known frequency distributions of TST readings of populations with and without LTBI, the latter including a fraction with non-specific tuberculin reactivity. RESULTS: Four group contact investigations were analyzed retrospectively with this tool. In two the graphed TST readings of contacts fell within the distribution of a population with LTBI, and suggested that transmission had occurred. In the other two, the graphed readings better fit the distribution of a population with non-specific tuberculin reactivity and suggested that transmission had not occurred. CONCLUSION: This simple tool to facilitate the determination of whether transmission of tuberculosis has occurred, and who should be offered treatment for LTBI in contact investigations of groups of people, deserves further study.

IGF-1 receptor mediates differentiation of primary cultures of mouse skeletal myoblasts.

Studies involving immortalized myoblasts suggested that insulin-like growth factors (IGFs) promote differentiation of skeletal muscle, but gene targeting experiments in mice did not provide support for this hypothesis. To address this discrepancy, we examined differentiation of primary cultures of mouse myoblasts. Differentiation was normally unaffected by addition of IGFs to the differentiation medium. However, when we interrupted IGF-mediated signaling, by incubating myoblasts with suramin or with a monoclonal antibody to the IGF-I receptor, differentiation was inhibited. Inhibition was reversed by exogenous IGF-I or IGF-II, but not by insulin. Differentiation was enhanced in myoblasts that were incubated with an inhibitor of the mitogen-activated protein kinase signaling pathway (PD098059) and such cells were responsive to exogenous IGF-I. Our results demonstrate that IGF action contributes to the differentiation of non-immortalized mouse myoblasts and that these cells represent a model system that can be experimentally manipulated to study the molecular events involved in skeletal muscle differentiation.

Treatment of isoniazid-resistant tuberculosis with isoniazid, rifampin, ethambutol, and pyrazinamide for 6 months.

SETTING: In 1992 the Seattle-King County Department of Public Health Tuberculosis Clinic began to treat patients with isoniazid-resistant tuberculosis with a regimen of isoniazid, rifampin, pyrazinamide, and ethambutol daily for 6 months. OBJECTIVE: To conduct a review of clinical and bacteriological outcomes of treatment for patients who received the four-drug, 6-month regimen for isoniazid-resistant tuberculosis. DESIGN: A retrospective review of medical records of TB cases meeting the study criteria, a Mycobacterium tuberculosis isolate resistant to isoniazid, and intent to treat with a 6-month course of isoniazid, rifampin, pyrazinamide, and ethambutol. RESULTS: Through December 1999, 44 consecutive patients with isoniazid-resistant, rifampin-susceptible tuberculosis were started on the four-drug, 6-month daily regimen. Among 42 patients followed until completion of therapy, three required changes in the regimen due to side effects. There was one case of drug-induced hepatotoxicity. Among 39 patients with pulmonary involvement, 37 converted sputum cultures from positive to negative within 2 months of starting treatment. There were no treatment failures. On passive follow-up of at least 2 years on all patients, two patients relapsed. The single patient with bacteriological relapse did not develop further drug resistance. CONCLUSION: The regimen of isoniazid, rifampin, pyrazinamide, and ethambutol given daily for 6 months produced successful outcomes when used in a public health tuberculosis clinic as routine therapy for isoniazid-resistant tuberculosis.

Zebrafish insulin-like growth factor-I receptor: molecular cloning and developmental expression.

The biological actions of the insulin-like growth factors (IGFs) are mediated primarily by the IGF-I receptor (IGF-IR), and the IGF family has been highly conserved throughout vertebrate evolution. In this study we report the isolation of a 3 kb cDNA clone for the zebrafish IGF-IR that includes the complete 3' untranslated region and polyA tail and mapping of the receptor gene to zebrafish linkage group 7. The open reading frame deduced from the cDNA sequence encompasses the juxtamembrane and protein tyrosine kinase portions of the receptor, and is 70 and 67% identical to the corresponding regions of the IGF-IRs of the turbot and Xenopus, respectively. By RT-PCR, zebrafish IGF-IR expression was detected from early blastula to early larval stages of development. Using whole mount in situ hybridization, IGF-IR expression was detected after gastrulation. Expression was evident in most tissues but was particularly evident in the tail, in eye and ear primordia and in the brain.

Divergent evolution in M6P/IGF2R imprinting from the Jurassic to the Quaternary.

M6P/IGF2R imprinting first appeared approximately 150 million years ago following the divergence of prototherian from therian mammals. Although M6P/IGF2R is clearly imprinted in opossums and rodents, its imprint status in humans remains ambiguous. It is also still unknown if M6P/IGF2R imprinting was an ancestral mammalian epigenotype or if it evolved convergently. We report herein that M6P/IGF2R is imprinted in Artiodactyla, as it is in Rodentia and Marsupialia, but that it is not imprinted in Scandentia, Dermoptera and Primates, including ringtail lemurs and humans. These results are most parsimonious with a single ancestral origin of M6P/IGF2R imprinting followed by a lineage-specific disappearance of M6P/IGF2R imprinting in Euarchonta. The absence of M6P/IGF2R imprinting in extant primates, due to its disappearance from the primate lineage over 75 million years ago, demonstrates that imprinting at this locus does not predispose to human disease. Moreover, the divergent evolution of M6P/IGF2R imprinting predicts that the success of in vitro embryo procedures such as cloning may be species dependent.

Monotreme IGF2 expression and ancestral origin of genomic imprinting.

IGF2 (insulin-like growth factor 2) and M6P/IGF2R (mannose 6-phosphate/insulin-like growth factor 2 receptor) are imprinted in marsupials and eutherians but not in birds. These results along with the absence of M6P/IGF2R imprinting in the egg-laying monotremes indicate that the parental imprinting of fetal growth-regulatory genes may be unique to viviparous mammals. In this investigation, we have cloned IGF2 from two monotreme mammals, the platypus and echidna, to further investigate the origin of imprinting. We report herein that like M6P/IGF2R, IGF2 is not imprinted in monotremes. Thus, although IGF2 encodes for a highly conserved growth factor in chordates, it is only imprinted in therian mammals. These findings support a concurrent origin of IGF2 and M6P/IGF2R imprinting in the late Jurassic/early Cretaceous period. The absence of imprinting in monotremes, despite apparent interparental conflicts over maternal-offspring exchange, argues that a fortuitous congruency of genetic and epigenetic events may have limited the phylogenetic breadth of genomic imprinting to therian mammals. J. Exp. Zool. (Mol. Dev. Evol.) 291:205-212, 2001.

Imprint status of M6P/IGF2R and IGF2 in chickens.

Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

Comparison testing of current (PPD-S1) and proposed (PPD-S2) reference tuberculin standards.

Since 1951, the tuberculin PPD-S1 has been used to standardize commercial PPD reagents and perform special tuberculin surveys. PPD-S1 is now in short supply and a new standard (PPD-S2) has been manufactured. To determine if PPD-S2 is equivalent and can replace PPD-S1, we conducted a double-blind clinical trial. Between May 14 and October 28, 1997, 69 subjects with a history of culture-proven tuberculosis (TB patients) and 1,189 subjects with a very low risk for TB infection were enrolled, received four skin tests (with PPD-S1, PPD-S2, and one each of the commercially available PPDs), and had reactions measured by two trained observers. Among the TB patients, we found statistically indistinguishable immunogenicity (mean reaction size +/- standard deviation): 15.6 +/- 6.6 mm for PPD-S1 and 14.8 +/- 5.6 mm for PPD-S2. Among low-risk subjects, the tests had equally high specificities (PPD-S1, 98.7% and PPD-S2, 98. 5%), using a 10-mm cutoff. The number of discordant (negative versus positive) interpretations for PPD-S2, assuming that low-risk subjects who had a >/= 10 mm reaction to PPD-S1 were truly infected, was low (0.5%) and indistinguishable from the rate of discordant interpretations of the same test when read by two different observers (0.8%). The study results indicate that PPD-S2 is qualified to be used as the new U.S. reference standard for PPD tuberculin.

Binding of 125I-insulin-like growth factor-II to cells cultured in fetal bovine serum: a complication.

Insulin-like growth factor II is an important fetal mitogen in mice and humans and its biological activity is regulated in a complex manner. The peptide interacts with three membrane-bound receptors, with a superfamily of insulin-like growth factor binding proteins and with the proteoglycan, glypican-3. Recently, the blood protein, vitronectin, has been identified as a novel insulin-like growth factor II-binding protein. Many studies have used cell lines maintained in fetal bovine serum to identify cell surface insulin-like growth factor II binding sites. We now describe a complication associated with the interpretation of such in vitro studies. Fetal bovine serum-derived vitronectin adheres very tightly to tissue culture dishes. When cells that have been maintained in fetal bovine serum are incubated with 125I-insulin-like growth factor II, a substantial fraction of the 125I-insulin-like growth factor II apparently associated with the cell surfaces may represent radioliogand bound by the fetal bovine serum-derived vitronectin. This may result in over-estimation of cell surface insulin-like growth factor II binding sites.

Community-wide implementation of targeted testing for and treatment of latent tuberculosis infection.

Treatment of latent infection due to Mycobacterium tuberculosis will likely increase in importance as a strategy to prevent tuberculosis in the United States. This review was undertaken to assess how targeted testing and treatment of latent tuberculosis infection are currently organized, with a focus on the extension of those services from public health clinics to other community sites. Targeted testing programs are now being implemented in primary care neighborhood clinics, syringe-exchange programs, jails, and teen health clinics. Organizational issues at those new sites include the need for a tracking system for clinical follow-up and for incentives to promote adherence. There is increasing experience with directly observed treatment of latent tuberculosis infection. Communities that receive large numbers of immigrants and refugees should prioritize the evaluation of those whose chest radiographs are suggestive of tuberculosis. Current studies continue to point out imperfections in the current tools, such as the tuberculin skin test and isoniazid. Finally, the advent of managed care, especially for Medicaid recipients, presents both opportunities and challenges for expansion of population-based preventive health services.