Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.

Genome screening in human systemic lupus erythematosus: results from a second Minnesota cohort and combined analyses of 187 sib-pair families.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a loss of immunologic tolerance to a multitude of self-antigens. Epidemiological data suggest an important role for genes in the etiology of lupus, and previous genetic studies have implicated the HLA locus, complement genes, and low-affinity IgG (Fcgamma) receptors in SLE pathogenesis. In an effort to identify new susceptibility loci for SLE, we recently reported the results of a genomewide microsatellite marker screen in 105 SLE sib-pair families. By using nonparametric methods, evidence for linkage was found in four intervals: 6p11-21 (near the HLA), 16q13, 14q21-23, and 20p12.3 (LOD scores >/=2.0), and weaker evidence in another nine regions. We now report the results of a second complete genome screen in a new cohort of 82 SLE sib-pair families. In the cohort 2 screen, the four best intervals were 7p22 (LOD score 2.87), 7q21 (LOD score 2.40), 10p13 (LOD score 2.24), and 7q36 (LOD score 2.15). Eight additional intervals were identified with LOD scores in the range 1.00-1.67. A combined analysis of MN cohorts 1 and 2 (187 sib-pair families) showed that markers in 6p11-p21 (D6S426, LOD score 4.19) and 16q13 (D16S415, LOD score 3.85) met the criteria for significant linkage. Three intervals (2p15, 7q36, and 1q42) had LOD scores in the range 1.92-2.06, and another 13 intervals had LOD scores in the range of 1.00-1.78 in the combined sample. These data, together with other available gene mapping results in SLE, are beginning to allow a prioritization of genomic intervals for gene discovery efforts in human SLE.

Molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral RNA persists through formation of a double-stranded complex without associated genomic mutations or evolution.

Enterovirus infection and persistence have been implicated in the pathogenesis of certain chronic muscle diseases. In vitro studies suggest that persistent enteroviruses mutate, evolving into forms that are less lytic and display altered tropism, but it is less clear whether these mechanisms operate in vivo. In this study, persistent coxsackievirus RNA from the muscle of mice afflicted with chronic inflammatory myopathy (CIM) was characterized and compared with RNA from a virus that had established a persistent infection of G8 mouse myoblasts for 30 passages in vitro. Competitive strand-specific reverse transcription-PCR and susceptibility to RNase I treatment revealed that plus- and minus-strand viral RNAs were present at nearly equivalent levels in muscle and that they persisted in a double-stranded conformation. All regions of the viral genome persisted and were amplified as a series of seven overlapping fragments. Restriction endonuclease fingerprinting coupled with sequencing indicated that there was no evolution of the viral genome associated with its persistence in muscle. This contrasted with the productive persistent infection that was established in myoblast cultures, where plus-strand RNA predominated and persistent virus developed distinct mutations. In vitro persistence proceeded by a carrier culture mechanism and was completely dependent on production of infectious virus, since persistent viral RNA was not detected in cultures subjected to antibody-mediated curing. These experiments demonstrate that persistence of coxsackievirus RNA in muscle is not facilitated by distinct genetic changes in the virus that give rise to replication-defective forms but occurs primarily through production of stable double-stranded RNA that is produced as the acute viral infection resolves. The data suggest a mechanism for coxsackievirus persistence in myofibers and perhaps other nondividing cells whereby cells that survive infection could harbor persistent viral RNA for extended times without producing detectable levels of infectious virus.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families.

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21-23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21-33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores >/=1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

Lactate stress test in the diagnosis of mitochondrial myopathy.

The aim of the study was to determine the sensitivity and specificity of the lactate stress test in the detection of mitochondrial myopathies. Thirty one healthy subjects, 10 patients with non-mitochondrial myopathy and 26 patients with mitochondrial myopathy underwent lactate stress testing at a standardized workload of 30 W during 15 min on a bicycle ergometer. Lactate was determined before the exercise (R1), 5, 10, 15 min after starting the exercise (S5, S10, S15) and 15 min after finishing the exercise (R2). A result was interpreted as pathologic if more than two of the five lactate values were above the corresponding upper reference limits. The upper reference limits for the venous lactate at R1, S5, S10, S15 and R2 were 1.9, 2.0, 2.1, 2.0 and 1.7 mmol/l respectively. The lactate stress test was pathologic in 1/10 of the non-mitochondrial myopathies and in 18/26 of the mitochondrial myopathies. The sensitivity of the lactate stress test was 69%. The specificity of the test was 90%. In conclusion, the lactate stress test proved to be helpful for evaluating the integrity of the oxidative metabolism in the majority of patients with mitochondrial myopathy.

The role of the clinical rheumatologist in the establishment of a large sibling pair resource for systemic lupus erythematosus.

OBJECTIVE: To recruit a large cohort of sibling pairs with systemic lupus erythematosus (SLE) as a clinical and biologic resource for genetic studies in SLE. METHODS: Complementary approaches were used to identify suitable families. The study was advertised in the newsletters of the Lupus Foundation of America and the Arthritis Foundation. Fliers were mailed to 250 clinical rheumatologists across the US, as well as to the local branches of the Lupus Foundation. All advertisements displayed a toll-free telephone number for interested patients to contact our group. Patients were then screened in a telephone interview by a university rheumatologist and their diagnosis was subsequently verified by telephone with the treating physician. Retrospective review of medical records was used to confirm the accuracy of the clinical data obtained by telephone interview. RESULTS: About 1400 subjects were screened by telephone over a 3 year period. After interviews with subjects and their physicians, 179 families were recruited in which at least 2 siblings have definite SLE. Based on the telephone interviews, a detailed clinical, demographic, and family history database was established for all patients in the study. Over 80% of the study subjects receive their SLE care from rheumatologists in clinical practice. CONCLUSION: We found that rheumatologists were reliable in confirming or excluding the diagnosis of SLE by telephone. Targeted patient advertising followed by physician-to-physician interviews is a time efficient and accurate method for recruiting patients with SLE for large genetic studies and may be applicable to the study of other rheumatologic conditions.

Coxsackievirus-induced chronic inflammatory myopathy: virus variants distinguish between acute cytopathic effects and pathogenesis of chronic disease.

Infection with the Tucson strain of coxsackievirus B1 (CVB1T) causes the development of chronic inflammatory myopathy (CIM) and hind limb weakness in susceptible strains of mice. In this study, a panel of six plaque-purified viruses exhibiting either small or large plaque phenotypes was derived from parental CVB1T and parental CVB1T that had been passaged through monkey kidney cells. All six variants caused similar acute histopathology in muscle, but three of four passaged viruses (AMP1, AMP2, and AMP3) did not induce CIM while the fourth (MP3) caused some hind limb weakness but without associated muscle inflammation. In contrast, both viruses (MP1 and MP2) isolated directly from the parental CVB1T stock were myopathic. Large plaque MP2 caused higher mortality and more rapid inhibition of host cell biosynthesis, but both MP1 and MP2 induced CIM that was comparable to that induced by parental CVB1T. Plaque size was a stable characteristic of the variants but did not correlate with their ability to induce CIM. Five of the six variants showed equivalent levels of replication in muscle, monkey kidney cells, and GB myoblasts while one, AMP3, was selectively impaired for replication. Receptor binding and virus-induced inhibition of host cell transcription and translation were not linked to myopathogenicity. Thus, most of the passaged variants are robust infectious viruses, suggesting that viral induction of CIM does not depend solely on cytopathogenicity during the acute infection.

Genetic determinants of susceptibility to coxsackievirus B1-induced chronic inflammatory myopathy: effects of host background and major histocompatibility complex genes.

Infection of outbred CD-1 mice with the Tucson strain of coxsackievirus B1 (CVB1T) leads to the development of chronic hind limb weakness and associated inflammatory muscle disease. Host factors that influence susceptibility have not been studied in this mouse model of chronic inflammatory myopathy (IM). Therefore, the pathogenesis was examined by using different inbred strains of mice. Initially, seven strains of mice with either the H-2d or H-2b major histocompatibility complex (MHC) haplotype were evaluated. All strains showed similar levels of acute mortality caused by viral infection, but chronic weakness or inflammation did not develop in two strains with the B6 background, regardless of their MHC haplotype. In susceptible mice, weakness was more likely to develop in the H-2d strains than in mice with the H-2b haplotype. Based on these results, H-2 congenic strains of the susceptible B10 background (C57BL/10 and B10.D2) and the resistant B6 background (C57BL/6 and B6.C-H2d) were examined in greater detail. During acute infection, the kinetics and degree of viral replication in hind limb muscle were similar among B6 and B10 strains. By 4 weeks after infection, more intense chronic muscle inflammation and pathology were observed in susceptible B10 mice of the H-2d haplotype than in those of the H-2b haplotype. Resistant B6 mice did not show signs of inflammation or calcification, but they did exhibit some myopathic features, including centralized nuclei and variations in myofiber size and shape. These changes were less common in resistant B6 mice than in B10 strains but were significant when compared with changes in uninfected controls. Viral RNA persistence and elevated titers of antiviral IgG were more prevalent in but not restricted to susceptible strains. These studies demonstrate that host background genes confer resistance to chronic IM but also that MHC genes influence disease severity. They also reveal that susceptibility to acute CVB1T infection is under different genetic control than that which mediates development of chronic post-viral IM.

Abnormal complement-mediated immune clearance in MRL-lpr/+ heterozygote mice: dose-dependent effect of the Fas antigen mutation.

We have previously demonstrated decreased complement-mediated clearance of IgG-opsonized erythrocytes in mice homozygous for the lpr mutation of the fas gene (BALB/c-lpr/lpr, C57BL/6-lpr/lpr, and MRL-lpr/lpr). To further test the hypothesis that the lpr mutation leads to a series of events resulting in selectively decreased complement-mediated immune clearance, in vivo clearance rate data were obtained from MRL-lpr/+F1, F2, and reciprocal backcross mice. Southern analysis of genomic DNA extracted from F2 and backcross mice was used to correlate the presence of the normal fas gene or the lpr mutation with normal or decreased complement-mediated clearance, respectively. Mean clearance rate constants for complement-dependent sequestration and phagocytosis were significantly decreased in the group of F1, F2, and backcross mice compared to control BALB/c mice (P < 0.0001). Data correlating clearance rate parameters from F2 and backcross mice with their respective genotype demonstrated a dose effect of the lpr mutation on abnormal complement-dependent sequestration and phagocytosis (r > 0.98), with heterozygote mice expressing mean values approximately half of those observed in +/+ homozygotes. These data demonstrate that the presence of the lpr mutation of the fas gene is strongly correlated in a dose-dependent manner with abnormal complement-mediated immune clearance. This clearance defect may be one mechanism through which the lpr mutation acts as an enhancer of autoimmune disease. Reduced clearance of immune complexes would increase the likelihood of tissue deposition and immune-mediated damage.

The alpha-D-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for beta-glucosidase activation.

A heteroglycan responsible for the binding of the enzyme beta-1,4-D-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungus Trichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated beta-1,4-D-glucosidase, beta-1,4-D-xylosidase and N-acetyl-beta-1,4-D-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear alpha-1,6-D-mannan. The mannan core obtained by acid degradation stimulated the beta-glucosidase activity by 90%. Several glycosidases from Aspergillus niger were also activated by the T. reesei heteroglycan. The beta-glucosidase of Trichoderma was activated by mannan from Saccharomyces cerevisiae to a comparable extent.

Saccharomyces species assignment by long range ribotyping.

Type strains of 10 genotypically distinct Saccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS region (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknown Saccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, misidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.

Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species.

The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose. The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis. However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis. The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results. RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

Duration of virus persistence and its relationship to inflammation in the chronic phase of coxsackievirus B1-induced murine polymyositis.

Mice infected with the Tucson strain of coxsackievirus B1 (CVB1T) develop chronic T cell-mediated polymyositis that is manifest as the acute infection resolves and is characterized by hindquarter weakness and muscle inflammation. This model system was used to study persistence of CVB1T RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR). For the most part, RNA persistence reflected the myotropic and neurotropic nature of the virus. At 1 month after infection, infectious virus was not detected in muscle, but persistent viral RNA was found in both skeletal and cardiac muscle, brain, and spinal cord. The kidney was weakly positive for viral RNA, whereas the liver and spleen were negative. Hindquarter muscle was assayed for persistent viral RNA at 1, 3, 6, 9, and 12 months after infection. In a few cases, persistent viral RNA was detected as late as 12 months after infection. The incidence of persistent viral RNA was high at 1 month after infection and gradually declined until, at 6 months and beyond, it was maintained in 3% to 12% of the muscles tested. Long-term viral RNA persistence was not more common in severely weak animals. However, the degree of hindquarter weakness that developed by 1 month was static thereafter and did not change over the 12-month study period. In contrast, separate experiments revealed that typical mononuclear cell (MNC) infiltration of muscle followed a time course similar to that of viral RNA persistence, peaking at 1 month and gradually resolving by 6 months. Infiltrating polymorphonuclear leukocytes (PMNs) and mast cells were present at 3 to 12 months after infection, signifying that some inflammatory activity remained. Other signs of myopathy that persisted for 12 months included a lack of muscle regeneration, variations in fiber size, and myofiber atrophy with increased perimysial and endomysial connective tissue. These results demonstrate that coxsackievirus RNA can persist in muscle for extended periods of time and are compatible with the idea that persistent virus is involved in maintaining the chronic MNC inflammation observed in murine polymyositis.