Patent Application: US-201514828415-A

Abstract:
describe herein is a novel catt - tetranucleotide repeat polymorphism at position − 817 of the human mif gene that functionally affects the activity of the macrophage inhibitory factor promoter in gene reporter assays . four genotypes are described which comprise 5 , 6 , 7 , or 8 - catt repeat units . of these , the 5 - catt allele has the lowest level of basal and stimulated mif promoter activity in vitro . the presence of the low expressing , 5 - catt repeat allele correlated with low disease severity in a cohort of rheumatoid arthritis patients . methods , compositions and apparatus for detecting this catt - tetranucleotide repeat polymorphism at position − 817 of the human mif gene , and for using same for assessing predisposition to severe inflammatory disease , are also disclosed .

Description:
the novel mif gene polymorphism identified herein is associated with reduced mif promoter activity , and the presence of this genotype in the homozygous state appears to be associated with a reduced risk of severe rheumatoid arthritis . mif has been shown to promote tnfα secretion and to enhance 1fnγ induced nitric oxide secretion from macrophages ( 8 ). in addition , mif is an important autocrine regulator of macrophage ( 8 ), t - cell ( 11 ) and fibroblast activation ( 18 ). these data have led to numerous investigations of the potential role for mif in chronic inflammatory conditions such as rheumatoid arthritis . mif protein levels circulate in higher levels in serum of rheumatoid arthritis patients and cellular mif expression is enhanced within the synovium ( 2 , 9 ). cultured synovial fibroblasts obtained from patients with rheumatoid arthritis secrete significant quantities of mif spontaneously in culture , and secretion increases further following pro - inflammatory stimulation ( 2 ). mif stimulation of rheumatoid synovial fibroblasts results in increased expression of matrix metalloproteinases ( 16 ), as well as the induction of phospholipase a 2 ( pla 2 ) and cox - 2 expression ( 29 ) immunoneutralization of mif activity in synoviocyte cultures also has been shown to inhibit il - iβ induced expression of cox - 2 and pla 2 mrna ( 29 ). the administration of a neutralizing anti - mif antibody also delays the onset and decreases the severity of type - ii collagen induced arthritis in mice ( 30 ) and profoundly inhibits the development of adjuvant - induced arthritis in rats ( 31 ). thus , there is considerable evidence implicating mif in the pathogenesis of inflammatory arthritis . disclosed herein is a significant association between patients that are homozygous for the low expressing , 5 - catt allele and less aggressive rheumatoid disease . only 1 / 79 ( 1 . 2 %) patients with severe rheumatoid arthritis inherited this genotype , compared with 101105 ( 9 . 5 %) of patients with milder , non - progressive disease . this suggests that a genetic predisposition to low expression of mif protects against persistent inflammation and / or joint destruction . it is unknown at present which transcription factors may be involved in modulating the transcriptional effects of the polymorphic region , but the 5 - catt allele shows reduced responses in vitro to both serum and forskolin stimulation as well as reduced basal activity . a catt repeated element also exists in the promoter of human granulocyte - macrophage colony - stimulating factor ( gm - csf ), and is required for promoter activity ( 32 , 33 ). it has been shown that the nuclear factor yy1 34 , and more recently the factors ap - 1 and sp - 1 , can form complexes with this region of the gm - csf promoter ( 35 ). whether any of these same factors also influence the activity of the mif catt repeat remains to be determined . the catt - repeat region within the mif gene contains several putative pit - 1 transcription factor binding sites . pit - 1 is a pituitary - specific transcription factor that is critical for the expression of pituitary hormones such as prolactin and growth hormone ( 36 ). the anterior pituitary gland is an important source of mif in rodents ( 3 ) and secretes mif in response to physiological or infective stress ( 37 ). corticotrophin - releasing factor ( crf ) also has been shown to be a potent inducer of mif expression in cultured pituitary cells . a recent fimctional analysis of the murine mif gene - promoter using rat pituitary cells and the pituitary cell line att - 20 demonstrated that crf - induced gene expression is dependent upon a camp responsive element binding protein ( 38 ). interestingly , reports of linkage of the crf locus to rheumatoid arthritis have recently appeared in the literature , and there is some evidence that the hypothalamic pituitary - adrenal ( hpa ) axis may play a role in the pathogenesis of rheumatoid arthritis in certain patients . patients with active rheumatoid arthritis have been shown to have abnormally low diurnal cortisol levels in the face of normal pituitary and adrenal function , suggesting a defect at the hypothalamic level ( 40 ). given mif &# 39 ; s capacity to counter - regulate glucocorticoid action within the immune system ( reviewed by bucala ( 1 )), the expression of mif by the anterior pituitary gland may be important to the development of inflammatory diseases such as rheumatoid arthritis . since the initiation of these studies , a − 173 * g / c single nucleotide polymorphism ( snp ) in the mif gene promoter has been reported by donn , et al . ( 41 ) and was shown to be associated with systemic - onset juvenile idiopathic arthritis ( systemic - onset jia ). the possession of at least one 173 * c allele was seen in 36 . 8 % of patients with systemic - onset jia compared to 20 . 3 % of the normal population ( 41 ). however , there is no information concerning the effect of this snp on gene expression . a preliminary analysis by the present inventors indicates that the 173 * c allele cannot explain the present association data or results of promoter assays ; indeed , there is no evidence of positive linkage disequilibrium between the 173 * c allele and the 5 - caat allele ( data not shown ). tnfα is considered to be a critical effector cytokine in rheumatoid arthritis , and anti - tnfα therapy has emerged to have high efficacy in the treatment of this disease ( 42 ). of note , there is a close relationship between mif and tnfα . mif appears to act as an important upstream regulator of tnfα expression . mif promotes secretion of tnfα from macrophages and overrides the ability of glucocorticoids to suppress macrophage tnfα production ( 43 ). immunoneutralization of mif also reduces circulating levels of tnfαa ( 3 ). in a clinical setting , the analysis of mif polymorphisms provides a prognosticator of disease severity , particularly in inflammatory diseases and more particularly in rheumatoid disease , and can assist in the selection of interventional therapy . the data herein also reaffirm the potential importance of mif as a therapeutic target in rheumatoid arthritis and possibly other inflammatory diseases . dna samples were obtained from the wichita rheumatic disease data bank and were representative of caucasian patients followed in a rheumatology practice since 1974 . the rheumatoid arthritis patients were divided into 2 groups using the following criteria : a ) severe ( n = 79 ); mean age at onset 55 years , mean disease duration of 13 years , mean larsen score rate of 4 . 0 , mean rf titer of 339 . 24 and a mean haq score of 1 . 36 . b ) mild ( n = 105 ); mean age at onset 45 years , mean disease duration of 15 years , mean larsen score of 1 . 0 , mean rf titer of 362 . 84 and a mean haq score of 0 . 93 . healthy caucasian volunteers provided genomic dna that was used as the normal control group ( n = 159 ). dna was extracted from whole blood using the g nome kit ( bio 101 inc ., ca , usa ) and from the buccal brushes using the pure gene kit ® ( gentra systems inc ., mn , usa ). the mif gene ( genbank accession number : l19686 , hereby incorporated in its entirety herein by reference ) is located on chromosome 22q11 . 2 ( 44 ). the gene is 2167 bp long and has 3 exons separated by 2 introns of 189 bp and 95 bp . four sets of primers were used to span the entire gene ( table 1 , below ). table 1 primer sequences and conditions for pcr of the mif gene annealing pcr pcr primer primer sequences temp special product set locations ( 5 ′- 3 ′) (° c .) conditions size set 1 mif - f (- 1074 ) tgcaggaaccaatacccatagg 58 . 1 654 bp ( seq . id no : 1 ) mif - r (- 421 ) tgcgtgagcttgtgtgtttgag ( seq . id no : 2 ) set 2 mif - f (- 441 ) tcaaacacacaagctcacgca 60 . 8 10 % dmso 445 bp ( seq . id no : 3 ) mif - r (+ 4 ) tggtcccgccttttgtg ( seq . id no : 4 ) set 3 mif - f (- 13 cacaaaaggcgggaccaca 62 . 3 25 % 7 - deaza 408 bp ( seq . id no : 5 ) gtp in 1 . 25 mif - r (+ 395 ) actgcgaggaaagggcg mm dntp ( seq . id no : 6 ) set 4 mif - f (+ 379 ) cgccctttcctcgcagt 10 % dmso 665 bp ( seq . id no : 7 ) mif - r (+ 1043 ) tagaatggaaagacactggg ( seq . id no : 8 ) the pcr reaction consisted of 1 × pcr buffer ii ( perkin elmer , ca , usa ), 20 ng dna , 1 . 5 mm mgcl 2 , 20 pmoles each of forward and reverse primers and 0 . 5 units of amplitaq gold ® polymerase ( perkin elmer - applied biosystems , ca , usa ). the dntp were used at a concentration of 0 . 2 mm except for set 3 , where the 0 . 2 mm dntp had 0 . 05 mm of 7 - deaza gtp in a 20 μl pcr reaction . the pcr conditions were as follows : 95 ° c ./ 12 min , followed by 40 cycles of 95 ° c ./ 30 sec , annealing temp ( table 1 )/ 30 sec , 72 ° c ./ 60 sec and 72 ° c ./ 10 min . the pcr products were resolved using a 1 % agarose gel stained with ethidium bromide . the pcr products from 6 normal controls and 6 rheumatoid arthritis patients were sequenced using the big dye terminator ® cycle sequencing ready reaction kit ( perkin elmer - applied biosystems ). the sequences from all four primer sets were compiled to represent the entire mif gene and were compared to analyze differences between the rheumatoid arthritis group and the normal controls . the forward primer from set 1 ( seq . id . no : 1 ) was used with the reverse primer mif - r − 728 ( 5 ′- aatggtaaactcggggac - 3 ′; seq . id no : 9 ). the reverse primer was fluorescently labeled with tet to allow detection of the pcr products using capillary electrophoresis ( 45 ). the pcr conditions were 1 × pcr buffer ii , 1 . 5 mm mgcl 2 , 0 . 2 mm dntp , 0 . 75 pmoles of each primer , 1 ng dna , 0 . 05 μl amplitaq gold ® polymerase in a 10 μl pcr reaction . the pcr cycling conditions used were the same as described above except for annealing conditions of 53 . 8 ° c ./ 30 sec . 1 μl of diluted pcr product was added to 12 μl of deionized formamide containing 0 . 5 μl gs - 500 tamra size standard ( perkin elmer - applied biosystems ). samples were denatured before being resolved using an abi 310 genetic analyzer ( perkin elmer - applied biosystems ). dna samples from homozygous individuals that previously had been fully sequenced were used as controls for the repeat sizes obtained by capillary electrophoresis . genomic dna obtained from the primary screening that contained the 5 , 6 , 7 , or 8 - catt tetranucleotide repeat polymorphism was used as a pcr template for initial cloning into the pcr2 . 1 - topo vector ( invitrogen , ca , usa ). the following primers were used to generate a 1173 - 1189 bp pcr product representing 1071 - 1087 bp of the upstream flanking region of the mif coding sequence plus the first 102 bp of exon i ( see fig1 ): after complete sequencing , the promoter region was excised from the pcr2 . 1 vector and cloned into the xhol / hindiii sites of the pgl3 - basic luciferase vector ( promega , wi , usa ). this vector contains the cdna encoding a modified version of firefly luciferase in the absence of eukaryotic enhancer or promoter elements . luciferase constructs directly regulated by the mif promoter , containing the 5 , 6 , 7 , or 8 - catt polymorphism , were generated . transient transfections were carried out using 3 μl fugene 6 ( roche , nj , usa ) and 1 μg of dna per well of a six well plate as per manufacturers directions . cell lines used included cos - 7 ( monkey kidney fibroblast ), a549 ( human lung epithelium ) and ccd - 19lu ( primary human lung fibroblast ). data were normalized in relation to an internal control of renilla luciferase that was regulated by the herpes simplex virus thymidine kinase promoter ( prl - tk vector — promega , wi , usa ). subsequently , each transfection consisted of 800 ng of test dna ( mif - promoter regulated luciferase gene ) combined with 200 ng of prl - tk control vector dna . luciferase assays were measured using a td - 20 / 20 luminometer ( turner designs , ca , usa ) and the dual luciferase reporter system ( promega , wi , usa ). basal promoter activity was determined by measuring luciferase activity 36 hours after transfection . in some cases , cells were stimulated for the last 20 hours of culture prior to measurement of promoter activity . the data were analyzed using genotyper ® 2 . 1 software ( perkin elmer - applied biosystems , ca , usa ). the relationship between the genotypes and disease status ( normal , mild or severe ) was examined using the chi - square test and fishers &# 39 ; exact test . gene reporter assays were repeated 3 to 10 times in duplicate . data are presented as mean ± stdev and compared by non - parametric mann - whitney u tests . significance was defined as p & lt ; 0 . 05 . genomic dna from six normal volunteers and six rheumatoid patients was utilized for full sequencing of the mif gene . due to the high gc content of this gene , the analysis was carried out in four sections . alignment of all twelve sequences identified a tetra - nucleotide catt repeat polymorphism in the upstream promoter region at position − 817 ( fig1 ). individuals having 5 , 6 , 7 or 8 - catt repeat alleles in their sequences were found . individuals were either heterozygous or homozygous for these alleles , although no 7 - catt homozygotes were found in the normal population and no 8 - catt homozygotes were found in either population studied . for rapid screening of the promoter polymorphism , a fluorescently labeled reverse primer that was proximal to the tetranucleotide repeat units was designed in order to amplify a smaller pcr fragment ( 340 - 352 bp ). this fragment then was analyzed using capillary electrophoresis on an abi310 genetic analyzer . the dna of individuals previously sequenced was used as a template to generate control dna fragments in order to correlate the fragment size observed on the abi310 analyzer with the number of catt repeats in the test samples . accordingly , the 4 pcr product sizes were 340 , 344 , 348 , and 352 bp in length , and these corresponded to five , six , seven , and eight - catt repeats , respectively . the genotypes observed were : 5 , 5 ; 5 , 6 ; 5 , 7 ; 6 , 6 ; 6 , 7 ; 7 , 7 ; 5 , 8 ; and 6 , 8 . the 8 , 8 genotype was not seen in either the normal ( n = 159 ) or patient ( n = 184 ) populations ; and the 7 , 7 genotype was not seen in the normal population , but was observed in one patient within the rheumatoid arthritis group . distribution of mif alleles in normal controls and rheumatoid arthritis patients . the distribution of the different mif alleles in normal controls , mild rheumatoid arthritis and severe rheumatoid arthritis patients are shown in table 2 , below . the number of individuals carrying at least one 5 - catt allele decreases from 50 . 31 % in the normal population to 31 . 65 % in the severe rheumatoid arthritis population ( table 2 ). the difference between the severe rheumatoid arthritis patients and controls is statistically significant ( p & lt ; 0 . 02 ). the cases and controls analyzed in this study were not closely matched for geographic and ethnic origin , hence the data must be interpreted with some caution . a comparison of specific genotypes between the mild and severe rheumatoid arthritis populations was therefore carried out , as shown in table 2 . the 5 , 5 genotype is observed in 9 . 5 % of the patients with mild rheumatoid arthritis , but is significantly decreased to 1 . 3 % in the patients with severe disease ( p = 0 . 0252 by fisher &# 39 ; s exact test ). these data indicate that a homozygous 5 - catt allele is protective for the development of severe disease . to investigate whether the catt repeat polymorphism was associated with functional regulation of mif expression , a gene reporter assay was developed and studied under defined conditions in vitro . gene reporter assays have been widely employed to study transcriptional regulation , or as readouts to monitor transcription factor ( 21 , 22 ). transfection of the mif promoter - regulated luciferase constructs into cos - 7 cells , a549 cells , and ccd - 19lu cells was associated with strong basal promoter activity , as indicated by high luciferase production , when compared to control vector ( pgl3 - basic ) ( fig2 and data not shown ). promoter activity was increased by forskolin ( an inducer of camp synthesis 23 ) and serum stimulation ( fig3 ), as well as phorbol ester stimulation ( data not shown ). in general , basal promoter activity was high in each of the cell lines tested when compared to negative ( empty pgl3 vector ) and positive ( prl - tk ) controls , and these data appeared to correlate with the high level of endogenous mif protein expression that was observed in these cell lines ( data not shown ). of note , in each of the cell lines tested , the 5 - catt repeat mif promoter construct showed significantly lower transcriptional activity when compared to the 6 , 7 , or 8 - catt repeat promoter constructs . the following documents are cited parenthetically by number in the specification above . 1 . bucala , “ mif rediscovered : cytokine , pituitary hormone , and glucocorticoid - 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1785 ( 2001 ). 42 . feldmann , et al ., “ anti - tnf - α alpha therapy of rheumatoid arthritis : what have we learned ?”, ann . rev . immunol ., 19 , 163 - 196 ( 1901 ). 43 . calandra , et al ., “ mif , a previously unrecognized macrophage cytokine , induces macrophages to secrete tnf - α and overcomes dexamethasone - suppression of tnf secretion ”, clinical research , 42 , a138 ( 1994 ). 44 . paralkar , et al ., “ cloning the human gene for macrophage migration inhibitory factor ( mif )”, genomics , 19 : 48 - 51 ( 1994 ). 45 . tsuda , et al ., “ separation of nucleotides by high - voltage capillary electrophoresis ”, j . appl . biochem ., 5 , 330 - 336 ( 1983 ). all patents , patent applications and publications mentioned hereinabove are hereby incorporated by reference in their entirety . while the foregoing invention has been described in some detail for purposes of clarity and understanding , it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims .