Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

The association of PTPN22 rs2476601 with juvenile idiopathic arthritis is specific to females.

A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. PTPN22 rs2476601 is associated with JIA and numerous other autoimmune diseases, and has been reported to show female-specific association with type 1 diabetes. We performed main effect and sex-stratified association analyses to determine whether a sex-specific association exists in JIA. As expected, rs2476601 was associated with JIA in our discovery (413 cases and 690 controls) and replication (1008 cases and 9284 controls) samples. Discovery sample sex-stratified analyses demonstrated an association specifically in females (odds ratio (OR)=2.35, 95% confidence interval (CI)=1.52-3.63, P=0.00011) but not males (OR=0.91, 95% CI=0.52-1.60, P=0.75). This was similarly observed in the replication sample. There was evidence for genotype-by-sex interaction (Pinteraction=0.009). The association between rs2476601 and JIA appears restricted to females, partly accounting for the predominance of females with this disease.

Adult onset distal and proximal myopathy with complete ophthalmoplegia associated with a novel de novo p.(Leu1877Pro) mutation in MYH2.

An MYH2 mutation p.(Glu706Lys) was originally described in a family with autosomal dominant inheritance, where the affected family members presented with multiple congenital contractures and ophthalmoplegia, progressing to a proximal myopathy in adulthood. Another patient with a dominant mutation p.(Leu1870Pro) was described, presenting as a congenital myopathy with ophthalmoplegia. Here, we present a patient with symptoms beginning at age 16 years, of prominent distal but also proximal weakness, bulbar involvement and ophthalmoplegia. Initially, clinically classified as oculopharyngodistal myopathy, the patient was found to carry a novel, de novo MYH2 mutation c.5630T>C p.(Leu1877Pro). This expands the phenotype of dominant MYH2 myopathies with the clinical phenotype overlapping the oculopharyngodistal myopathy spectrum.

Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens.

Members of the genus Bacillus produce a wide variety of antimicrobial compounds. Cyclic lipopeptides (CLP) produced by Bacillus subtilis strains have been shown to protect host plants from a numbers of pathogens. The representative families of these CLP (surfactins, fengycins, and iturins) share a polypeptide ring linked to a lipid tail of varying length. CLP provide plant protection through a variety of unique mechanisms. Members of the surfactin and fengycin families elicit induced systemic resistance in certain host plants, and they also function by directly affecting the biological membranes of bacterial and fungal pathogens, mainly resulting in membrane pore formation. Specific pore forming mechanisms differ between CLP families, causing differential activities. CLP also may aid in enhanced B. subtilis colonization of the plant environment in addition to potentially preventing the adhesion of competitive microorganisms. Several recent studies have highlighted the control of plant pathogens by CLP-producing B. subtilis strains. Strong ecological advantages through multifaceted activities of CLP provide these strains with immense promise in controlling pathogens in a variety of plant ecosystems.

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

Gene dosage is a mechanism for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy in humans, characterized electrophysiologically by decreased nerve conduction velocities (NCVs). CMT1A is associated with a large submicroscopic DNA duplication in proximal 17p. In this report we demonstrate that a patient with a cytogenetically visible duplication, dup(17)(p11.2p12), has decreased NCV. Molecular analysis demonstrated this patient was duplicated for all the DNA markers duplicated in CMT1A as well as markers both proximal and distal to the CMT1A duplication. These data support the hypothesis that the CMT1A phenotype can result from a gene dosage effect.

Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12.

Bacillus subtilis strains are known to produce a vast array of antimicrobial compounds. However, some compounds remain to be identified. Disk assays performed in vitro with Bacillus subtilis CU12 showed a significant reduction in mycelial growth of Alternaria solani, Botrytis cinerea, Fusarium sambucinum, and Pythium sulcatum. Crude B. subtilis culture filtrates were subsequently extracted with ethyl acetate and butanol. A bioassay guided purification procedure revealed the presence of one major antifungal compound in the butanol extract. Purification of the compound was performed using a reverse-phase C18 solid phase extraction (SPE) cartridge and flash column chromatography. NMR data showed that the main antimicrobial compound was a cyclic dimer of 3-hydroxypropionaldehyde (HPA). This study demonstrated the antimicrobial activity of B. subtilis strain CU12 against phytopathogenic microorganisms is mediated at least in part by the production of HPA. It also suggests that this B. subtilis strain could be effective at controlling pathogens through protection of its ecological niche by antibiosis.

What causes AIS? Ask the genome!

The most common developmental disorder of the spine is scoliosis, a rotated, lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied pathogenic understanding in part due to its genetic complexity, and to the lack of well-defined animal models. The disease is also remarkable in its sexual dimorphism, where girls are at more than five times greater risk of progressive deformity than boys. Breakthroughs have come from recent genome wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts. Post-hoc gene set and pathway-level analyses of genetic datasets have highlighted a role for cartilage biogenesis and the development of the intervertebral disc (IVD) in disease susceptibility. Moreover, next generation sequencing in AIS families, as well as modeling in vertebrate systems, has revealed that rare deficiencies in proteins of the cartilaginous extracellular matrix (ECM) collectively contribute to AIS. Thus, as in a jigsaw puzzle, the pieces coming together from multiple biologic studies suggest that deficiencies in the structural integrity and homeostasis of spinal cartilages are culprits in AIS susceptibility. Here, we update progress in understanding the genetic, biochemical, and cellular determinants of AIS. We also suggest a molecular model in which interaction of the hormonal environment with genetic susceptibility may increase risk of this common disorder of childhood.

Facilitation of brain self-stimulation by central administration of norepinephrine.

Rats with electrodes implanted in the medial forebrain bundle stimulated their own brains at sharply reduced rates after systemic administration of disulfiram or intraventricular administration of diethyldithiocarbamate. Both drugs inhibit dopamine-beta-hydroxylase, the enzyme responsible for the final step in the biosynthesis of norepinephrine. The suppressed behavior was reinstated by intraventricular injections of 1-norepinephrine, but not by injection of its biologically inactive isomer, d-norepinephrine. Intraventricular administration of dopamine and serotonin did not restore self-stimulation. The rewarding effect of medial forebrain bundle stimulation may depend on the availability of norepinephrine as a transmitter, but not on dopamine or serotonin.

Possible etiology of schizophrenia: progressive damage to the noradrenergic reward system by 6-hydroxydopamine.

Single or repeated intraventricular injections of 6-hydroxydopamine caused marked and long-lasting deficits in brain self-stimulation and other rewarded behaviors in the rat. The behavioral deficits, as well as the depletion of brain norepinephrine induced by 6-hydroxydopamine, were prevented by prior treatment with chlorpromazine. Episodic or continuous formation of endogenous 6-hydroxydopamine in man as a result of a genetically determined enzymatic error could selectively damage the binding capacity and, eventually, the structural integrity of the noradrenergic reward mechanism. Such damage might cause the fundamental symptoms and long-term downhill course of schizophrenia.

Dopamine-beta-hydroxylase deficits in the brains of schizophrenic patients.

Postmortem brain specimens from 18 schizophrenic patients and 12 normal controls were assayed for dopamine-beta-hydroxylase (DBH), the enzyme responsible for the final step in norepinephrine biosynthesis. There was a significant reduction in the DBH activity of the schizophrenic group in all brain regions examined. Enzyme deficits in hippocampus and diencephalon were somewhat larger than that in pons-medulla. Since various extraneous factors, such as non-specific deterioration, drug treatment, duration of hospitalization, cause of death, sex, and age could be ruled out, the deficits in DBH mnay be associated with the schizophrenic disease process. These findings are consistent with the hypothesis that noradrenergic "reward" pathways are damaged in schizophrenia.

Norepinephrine: reversal of anorexia in rats with lateral hypothalamic damage.

Injection of norepinephrine in the lateral ventricles of rats recovering from lateral hypothalamic anorexia caused immediate feeding and, frequently, overeating. Intraventricular administration of the alpha-noradrenergic blocker, phentolamine, suppressed feeding in both normal rats and rats that had recovered from lateral hypothalamic lesions. Feeding is reinforced by ascending medial forebrain bundle fibers that form alpha-noradrenergic synapses in the hypothalamus and forebrain. Damage to these fibers suppresses feeding by reducing noradrenergic transmission and, hence, the rewarding value of food. Recovery of feeding after hypothalamic lesions coincides with the recovery of noradrenergic reward function.

Nerve growth factor: enhanced recovery of feeding after hypothalamic damage.

A single intraventricular injection of nerve growth factor (NGF). given at the time of brain damage, facilitated the course of recovery from the lateral hypothalamic anorexic syndrome in male rats. In the second and third weeks after the trauma, NGF-treated rats ate more food, regained body weight more rapidly, and fed more vigorously in response to intraventricular administration of norepinephrine than untreated controls. After full recovery, rats that had been treated with NGF were resistant to reinstatement of the hypothalamic syndrome by 6-hydroxydopamine. NGF may facilitate behavioral recovery by promoting the development of supersensitivity to norepinephrine and possibly also by stimulating the growth of regenerating noradrenergic neurons in the brain.

Benzodiazepines: anxiety-reducing activity by reduction of serotonin turnover in the brain.

The anxiety-reducing effects of minor tranquilizers in the rat conflict test were mimicked by serotonin antagonists and by p-chlorophenylalanine, an inhibitor of serotonin synthesis; the depressant effects of the minor tranquilizers were mimicked by norepinephrine antagonists. Intraventricular injections of serotonin led to a suppression of behavior, and also antagonized the anxiety-reducing action of benzodiazeprines. Intraventricular injections of norepinephrine led to a release of punished behavior from suppression, and also antagonized the depressant action of benzodiazepines. The anxiety-reducing activity, and the decrease in serotonin turnover induced by benzodiazepines, were maintained over repeated doses, whereas depressant activity, and the decrease induced in norepinephrine turnover, both rapidly underwent tolerance. Tranquilizers may exert their anxiety-reducing effects by a reduction of serotonin activity in a behaviorally suppressive punishment system, and they may exert their depressant effects by a reduction of norepinephrine activity in a behaviorally facilitatory reward system.

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.

Mutations responsible for Larsen syndrome cluster in the FLNB protein.

BACKGROUND: A gene for Larsen syndrome was recently described, and mutations were reported in five cases. OBJECTIVE: To test whether mutations in this gene, FLNB, could explain the disease in our independent collection of sporadic and dominant Larsen syndrome cases; and to test whether mutations occurred in a non-random pattern. RESULTS: Missense mutations were found in each of five cases. Four of the five were new; one was reported in a sporadic case in the original Larsen syndrome study of five cases. All mutations from the two studies were compiled. Clustered mutations were observed within three filamin B protein domains: the calponin homology 2 domain, repeat 14, and repeat 15. This suggested that as few as five (of the total of 46) coding exons of FLNB could be screened to detect Larsen syndrome mutations. Four of these exons were screened in a sixth (sporadic) case and a previously reported G1691S substitution mutation detected. CONCLUSIONS: Mutations in FLNB may be responsible for all cases of Larsen syndrome. They appear to occur in specific functional domains of the filamin B protein. This should simplify diagnostic screening of the FLNB gene. Analyses in larger patient series are warranted to quantify this. The study confirmed the extreme variability in clinical presentation and the presence of unaffected carriers. A molecular screen would be valuable for diagnosis and genetic counselling.

Insulin binding and response in erythroblastic leukemic cells.

Mammalian erythrocytes have been shown to bind, but not to respond to, physiologic doses of insulin. Insulin binding was studied in normal rat erythrocytes and erythroblastic leukemic (EBL) cells by standard 125I-insulin competitive binding assays. EBL cells exhibited marked insulin degradation, which was time, temperature, and concentration dependent and was mediated by both cell-bound and soluble enzymes. Bacitracin or bovine serum albumin was used to inhibit such degradation in a dose-dependent fashion to allow meaningful data analysis. Insulin binding studies showed a greater than 10-fold increase of specific binding to EBL cells compared with erythrocytes. Scatchard analysis was consistent with increases predominantly in the number of receptors on EBL cells. Concordant with increased insulin binding, EBL cells demonstrated increased transport of alpha-aminolsobutyric acid and increased incorporation of uridine into ribonucleic acid in response to physiologic doses of insulin (100 microunits/ml). It can be concluded that EBL cells may serve as useful models of erythroblasts to explore the relationships between insulin binding, response, and cell maturation.

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.

Gonococcal arthritis in an era of increasing penicillin resistance. Presentations and outcomes in 41 recent cases (1985-1991).

BACKGROUND: To assess the impact of recent reports of disseminated gonococcal infection caused by penicillin-resistant organisms, we reviewed the presenting features, clinical course, and outcomes of a group of patients with gonococcal arthritis treated in recent years. METHODS: We reviewed the records of all cases of acute arthritis associated with a culture positive for Neisseria gonorrhoeae at our institution from July 1985 through December 1991. RESULTS: Forty-one cases were identified. Patients included 34 women and 38 blacks; the mean age was 22.6 years. Duration of symptoms averaged 4.8 days at presentation. Other features included migratory arthralgias (n = 27), urogenital symptoms or signs (n = 26), fever (n = 21), and skin lesions (n = 16). Comorbid conditions included intravenous drug use (n = 8) and systemic lupus erythematosus (n = 3). The knee was the most commonly affected joint. Positive culture results were obtained from 32 urogenital samples (86%), 14 synovial fluid samples (44%), seven rectal samples (39%), four blood samples (12%), and two throat samples (7%). All synovial fluid samples with positive culture results had white blood cell counts higher than 20.0 x 10(9)/L. Response to therapy with penicillin and/or ceftriaxone was prompt, and mean duration of hospitalization was 5.8 days. Patients who required longer hospitalization had a higher mean erythrocyte sedimentation rate and higher frequencies of positive synovial fluid culture results and comorbid conditions. Penicillin sensitivity could be determined in 30 patients on the basis of clinical response or in vitro testing. Among these patients, two cases of penicillin-resistant organisms were identified, one beta-lactamase positive and one beta-lactamase negative. CONCLUSIONS: The clinical features of patients with gonococcal arthritis have changed very little since the last large reported series over a decade ago. Underlying conditions appear to be more common, but response to antibiotic therapy and eventual outcome remain excellent. The finding of penicillin-resistant organisms in at least 5% of patients reinforces recent recommendations that third-generation cephalosporin agents be used as initial therapy for disseminated gonococcal infections until drug susceptibilities are known.

Presenting features and outcomes in patients undergoing temporal artery biopsy. A review of 98 patients.

BACKGROUND: Although temporal arteritis is a well-recognized syndrome, controversy still exists regarding the optimal approach to diagnosis and treatment of this condition. We undertook this review to further define the spectrum of presenting features and outcomes of patients undergoing temporal artery biopsy. METHODS: We reviewed the records of all patients undergoing temporal artery biopsy over a 5-year period. Presenting features were compared in biopsy-positive and biopsy-negative patients. In patients with positive biopsy specimens, treatment regimens, disease, treatment-related morbidity, and outcomes were recorded. Alternative diagnoses and therapy were reviewed in biopsy-negative patients. RESULTS: Of 98 patients, 30 had positive and 68 had negative biopsy specimens. Biopsy-positive patients had an increased incidence of headache (93% vs 62%), jaw claudication (50% vs 18%), and prior polymyalgia rheumatica (23% vs 3%), but the sensitivity and specificity of these indicators were relatively low. Other clinical and laboratory parameters, including prior steroids and erythrocyte sedimentation rate, were similar between the two groups. In 30 patients with positive biopsy specimens, response to initial high-dose steroid was excellent. Serious manifestations after initial treatment were not seen, but mild flares were common after 1 year of therapy. Steroid-related morbidity was common, and steroids were seldom discontinued (0/22 patients at 1 year, 6/19 patients at 2 years, 5/11 patients at 3 years). In 68 patients with negative biopsy specimens, alternative diagnoses included neurologic diseases (15 patients), "pure" polymyalgia rheumatica (14 patients), and other inflammatory rheumatologic diseases (10 patients). Fourteen patients with negative biopsy specimens were treated for temporal arteritis, and were similar to biopsy-positive patients. CONCLUSIONS: Temporal arteritis remains a challenging condition to diagnose and to treat. Presenting features are seldom helpful in predicting biopsy results. Initial treatment is effective but frequently toxic. Although late disease-related complications are rare, most patients continue to take long-term low-dose steroid therapy.