Patent Application: US-201514844899-A

Abstract:
a method for measuring blood levels of β cell dna that is released upon β cell death by using a quantitative probe technology to detect amplified methylated and demethylated forms of the insulin gene dna , representing normal tissue and β cell specific origin , respectively . using probes permits the sensitive and specific identification of demethylated insulin dna patterns that are present only in β cells . the method offers a bioassay for detecting β cell loss in diabetes , useful for screening of prediabetes , monitoring of disease progression , and selection and monitoring of therapies . the technique finds potential use in both type i and type ii diabetes , as well as gestational diabetes .

Description:
in various embodiments , the present technology substantially isolates nucleic acids from a sample of body fluid , for example blood plasma , saliva , spinal fluid , lymph fluid , synovial fluid , or tears , for example . various dna extraction , isolation and purification technologies can be used , for example as taught in u . s . pat . nos . 4 , 935 , 342 , 5 , 990 , 301 , 6 , 020 , 124 , 7 , 241 , 596 , 6 , 485 , 903 , 6 , 214 , 979 , re . 39 , 920 each of which is expressly incorporated herein by reference in its entirety . an anion exchange material may be selected and employed which effectively adsorbs the target nucleic acids or protein complexes thereof . for example , commercially available anion exchange materials may be employed . either strong or weak anion exchangers may be employed . a preferred weak exchanger can be one in which primary , secondary , or tertiary amine groups ( i . e ., protonatable amines ) provide the exchange sites . the strong base anion exchanger has quaternary ammonium groups ( i . e ., not protonatable and always positively charged ) as the exchange sites . both exchangers can be selected in relation to their respective absorption and elution ionic strengths and / or ph for the nucleic acid being separated . purification by anion exchange chromatography is described in u . s . pat . no . 5 , 057 , 426 ( see also ep 0 268 946 b1 ), expressly incorporated by reference herein in its entirety . the material which is commercially available under the designation q - sepharose ™ ( ge healthcare ) is a particularly suitable . q - sepharose ™, can be a strong anion exchanger based on a highly cross - linked , bead formed 6 % agarose matrix , with a mean particle size of 90 μm . the q - sepharose ™ can be stable in all commonly used aqueous buffers with the recommended ph of 2 - 12 and recommended working flow rate of 300 - 500 cm / h . in other preferred embodiments , the anion - exchange medium can be selected from sepharose - based quaternary ammonium anion exchange medium such as q - filters or q - resin . the chromatographic support material for the anion charge used in the instant methods can be a modified porous inorganic material . as inorganic support materials , there may be used materials such as silica gel , diatomaceous earth , glass , aluminum oxides , titanium oxides , zirconium oxides , hydroxyapatite , and as organic support materials , such as dextran , agarose , acrylic amide , polystyrene resins , or copolymers of the monomeric building blocks of the polymers mentioned . the nucleic acids can also be purified by anion exchange materials based on polystyrene / dvb , such as poros 20 for medium pressure chromatography , poros ™ 50 hq , of the firm of bioperseptive , cambridge , u . s . a ., or over deae sepharose ™ deae sephadex ™ of the firm of pharmacia , sweden ; deae spherodex ™, deae spherosil ™, of the firm of biosepra , france . a body fluid sample , such as blood plasma or saliva , containing nucleic acids or their proteinous complexes , is applied to the selected anion exchange material , and the nucleic acids or their complexes become adsorbed to the column material . the contact and subsequent adsorption onto the resin can take place by simple mixing of the anion exchange media with the body fluid , with the optional addition of a solvent , buffer or other diluent , in a suitable sample container such as a glass or plastic tube , or vessel commonly used for handling biological specimens . this simple mixing referred to as batch processing , can be allowed to take place for a period of time sufficiently long enough to allow for binding of the nucleoprotein to the media , preferably 10 to 40 min . the media / complex can then be separated from the remainder of the sample / liquid by decanting , centrifugation , filtration or other mechanical means . the anion exchange material can optionally be washed with an aqueous solution of a salt at which the nucleic acids remain bound to the anion exchange material , the washing being of sufficient volume and ionic strength to wash the non - binding or weakly binding components through the anion - exchange material . in some embodiments , the resin can be washed with 2 × ssc ( 300 mm nacl / 30 mm sodium citrate ( ph 7 . 0 ). preferred ranges of the salt solutions are 300 - 600 nm nacl / 30 mm sodium citrate ( ph 7 . 0 ). the resin may alternately be washed with 300 - 600 mm licl / 10 mm naoac ( ph 5 . 2 ). the bound nucleic acids may then be eluted by passing an aqueous solution through the anion exchange material of increasing ionic strength to remove in succession proteins that are not bound or are weakly bound to the anion - exchange material and the nucleic acids of increasing molecular weight from the column . both proteins and high and low molecular weight nucleic acids ( as low as 10 base pairs ) can be selectively eluted from the resin stepwise with the salt solution of concentrations from 300 mm to 2 . 0 m of nacl and finally with 2 . 0 m guanidine isothiocyanate . licl solutions in the concentration range of 300 mm to 2 . 0 m of licl may also be used for stepwise elution . the nucleic acids isolated may be in double - stranded or single - stranded form . the body fluid can be pre - filtered through a membrane and supplemented with 10 mm edta ( ph 8 . 0 ) and 10 mm tris - hcl ( ph 8 . 0 ) prior to adsorption onto the anion - exchange medium . commercial sources for filtration devices include pall - filtron ( northborough , mass . ), millipore ( bedford , mass . ), and amicon ( danvers , mass .). filtration devices which may be used are , for example , a flat plate device , spiral wound cartridge , hollow fiber , tubular or single sheet device , open - channel device , etc . the surface area of the filtration membrane used can depend on the amount of nucleic acid to be purified . the membrane may be of a low - binding material to minimize adsorptive losses and is preferably durable , cleanable , and chemically compatible with the buffers to be used . a number of suitable membranes are commercially available , including , e . g ., cellulose acetate , polysulfone , polyethersulfone , and polyvinylidene difluoride . preferably , the membrane material is polysulfone or polyethersulfone . the body fluid , for example blood plasma or saliva , can be supplemented with edta and tris - hcl buffer ( ph 8 . 0 ) and digested with proteinases , such as for example proteinase k , prior to adsorption onto the anion exchange medium . the anion - exchange medium can be immobilized on an individualized carrier such as a column , cartridge or portable filtering system which can be used for transport or storage of the medium / nucleoprotein bound complex . the nucleic acid / anion exchange may be maintained in storage for up to 3 weeks . a kit may be provided with a solid carrier capable of adsorbing the nucleic acids containing in a sample of a body fluid , for example blood plasma or saliva . the kit may also contain other components for example , reagents , in concentrated or final dilution form , chromatographic materials for the separation of the nucleic acids , aqueous solutions ( buffers , optionally also in concentrated form for final adjusting by the user ) or chromatographic materials for desalting nucleic acids which have been eluted with sodium chloride . the kit may also contain additional materials for purifying nucleic acids , for example , inorganic and / or organic carriers and optionally solutions , excipients and / or accessories . such agents are known and are commercially available . for solid phase nucleic acid isolation methods , many solid supports have been used including membrane filters , magnetic beads , metal oxides , and latex particles . widely used solid supports include silica - based particles ( see , e . g ., u . s . pub . pat . app . 2007 / 0043216 ( bair jr ., et al . ); u . s . pat . no . 5 , 234 , 809 ( boom et al . ); wo 95 / 01359 ( colpan et al . ); u . s . pat . no . 5 , 405 , 951 ( woodard ); wo 95 / 02049 ( jones ); wo 92 / 07863 ( qiagen gmbh ), each of which is expressly incorporated herein by reference ). inorganic components of carriers may be , for example , porous or non - porous metal oxides or mixed metal oxides , e . g . aluminum oxide , titanium dioxide , iron oxide or zirconium dioxide , silica gels , materials based on glass , e . g . modified or unmodified glass particles or ground glass , quartz , zeolite or mixtures of one or more of the above - mentioned substances . on the other hand , the carrier may also contain organic ingredients which may be selected , for example , from latex particles optionally modified with functional groups , synthetic polymers such as polyethylene , polypropylene , polyvinylidene fluoride , particularly ultra high molecular polyethylene or hd - polyethylene , or mixtures of one or more of the above - mentioned substances . in addition , the reagent kit may also contain excipients such as , for example , a protease such as proteinase k , or enzymes and other agents for manipulating nucleic acids , e . g . at least one amplification primer , and enzymes suitable for amplifying nucleic acids , e . g . dnase , a nucleic acid polymerase and / or at least one restriction endonuclease . alternately , a commercial polymerase chain reaction kit may be used to amplify the dna samples , as discussed below . dna is subject to degradation by dnases present in bodily fluids , such as saliva . thus , in certain embodiments , it is advantageous to inhibit dnase activity to prevent or reduce the degradation of dna so that sufficiently large sequences are available for detection . after collection , the sample may be treated using one or more methods of inhibiting dnase activity , such as use of ethylenediaminetetraacetic acid ( edta ), guanidine - hcl , gitc ( guanidine isothiocyanate ), n - lauroylsarcosine , na - dodecylsulphate ( sds ), high salt concentration and heat inactivation of dnase . after collection , the sample may be treated with an adsorbent that traps dna , after which the adsorbent is removed from the sample , rinsed and treated to release the trapped dna for detection and analysis . this not only isolates dna from the sample , but , some adsorbents , such as hybond ™ n membranes ( amersham pharmacia biotech ltd ., piscataway , n . j .) protect the dna from degradation by dnase activity . in some cases , the amount of dna in a sample is limited . therefore , for certain applications , sensitivity of detection may be increased by known methods . where dna is present in minute amounts , larger samples can be collected and thereafter concentrated such as by butanol concentration or concentration using sephadex ™ g - 25 ( pharmacia biotech , inc ., piscataway n . j .). once obtained , the bodily fluid derived dna may be used as an alternate to serum - derived dna as discussed below . since the technology is ratiometric , it is dependent not on the absolute quantity of dna available , but the proportional relationships of the methylated and unmethylated portions . in general , the disposition of these types in the various body fluids is not believed to be highly dependent on the fluid type , and calibration techniques can be used to account for persistent and predictable differences in the fluid methylated / unmethylated ratios . dna from serum samples was purified using the qiagen qiaamp dna blood kit following the manufacturer - recommended protocol . synthetic unmethylated and methylated dna was purchased from zymo research . dna was then subjected to bisulfite treatment and purified on a dna binding column to remove excessive bisulfite reagent using the zymo ez dna methylation kit . a methylation - independent reaction was carried out to increase the dna template for pcr analysis . for the reaction , bisulfite - treated dna template was added to zymo taq premix . the amplification proceeded for , e . g ., 50 cycles . the pcr products were excised from a 3 % agarose gel . negative controls without dna did not yield products in the first - step reaction . pcr products obtained using methylation - independent primers were purified using a qiagen pcr purification kit . methylation - specific dna probes are used for the detection of β cell derived dna . these probes are able to quantitatively and sensitively detect circulation demethylated and methylated dna from a β cell and a non - β cell origin , respectively . the new probes replace the previously published methylation specific primers ( see akirav e m , lebastchi j , galvan e m , henegariu o , akirav m , ablamunits v , lizardi p m , and herold k c . detection of β cell death in diabetes using differentially methylated circulating dna . pnas , 2011 , proceedings of the national academy of sciences , 2011 , november : 108 ( 19018 - 23 ), expressly incorporated herein by reference , hereinafter akirav et al . ( 2011 ). see also husseiny m i , kuroda a , kaye a n , nair i , kandeel f , et al . ( 2012 ) development of a quantitative methylation - specific polymerase chain reaction method for monitoring beta cell death in type 1 diabetes . plos one 7 ( 10 ): e47942 . doi : 10 . 1371 / journal . pone . 0047942 , expressly incorporated herein by reference ), which presented with a relatively low specificity ( i . e . demethylated primers detected methylated dna and vice versa ). low specificity negatively impacts assay sensitivity by decrease detection limits of β cell derived demethylated dna . low dna levels are presumably present during early β cell loss , such as prediabetes . see , u . s . pat . no . 6 , 150 , 097 , expressly incorporated herein by reference . the overall procedure for the detection of circulating β cell dna is depicted in fig1 . the steps leading to the use of probes are identical with those described in akirav et al . ( 2011 ), which discloses the use of methylation - specific primers ( and not probes ) to detect β cell derived dna . the primers were able to detect demethylated and methylated dna from a β cell and a non - β cell origin , respectively . while useful , these primers had a relatively low specificity whereby demethylated primers detected methylated dna and vice versa . low specificity reduced assay sensitivity as it impaired the ability to detect very low levels of β cell - derived dna , such as in the condition of early β cell loss and pre - diabetes . dna from serum samples was purified using the qiagen qiaamp dna blood kit following the manufacturer - recommended protocol . synthetic unmethylated and methylated dna was purchased from millipore . purified dna was quantitated using a nanodrop 2000 spectrophotometer . dna was then subjected to bisulfite treatment and purified on a dna binding column to remove excessive bisulfite reagent using the zymo ez dna methylation kit . the present method , in contrast , uses probe dna that offers a significant improvement in sensitivity over the primers used in the prior art discussed above . that is , probe dna allows for a highly specific recognition of two demethylated sites in the insulin gene . this tends to eliminate false positive readings and thus provides increased assay specificity and sensitivity . the following is used as probe for the detection of circulating dna in the assay according to the present method : a methylation - independent reaction was carried out to increase the dna template for pcr analysis . for the reaction , bisulfite - treated dna template was added to zymotaq ™ premix ( see , www . zymoresearch . com / protein / enzymes / zymotaq - dna - polymerase , expressly incorporated herein by reference .) the following pcr primers are used to amplify the human insulin position 2122220 - 2121985 on chromosome 11 , grch37 . p10 , october 2012 ): using the forward and reverse primers , pcr was conducted the pcr products were excised from a 3 % agarose gel . the pcr product ( or amplicon ) is detect by methylation status specific probes as follows : a ) probes for the detection of methylated insulin dna ( i . e ., dna not derived from a β cell )( alternates ): b ) probes for the detection of demethylated insulin dna ( i . e ., dna derived from a β cell )( alternates ): in various embodiments , the methylation status - specific probes are conjugated with 6 - carboxyfluorescein , abbreviated as fam , thus permitting quantitative detection . see , en . wikipedia . org / wiki / taqman , expressly incorporated herein by reference . other technologies may be used i conjunction with the present method ; see , u . s . pat . nos . 6 , 103 , 476 , 8 , 247 , 171 , 8 , 211 , 644 , 8 , 133 , 984 , 8 , 093 , 003 , 8 , 071 , 734 , 7 , 972 , 786 , 7 , 968 , 289 , 7 , 892 , 741 , 7 , 847 , 076 , 7 , 842 , 811 7 , 803 , 528 , 7 , 776 , 529 , 7 , 662 , 550 , 7 , 632 , 642 , 7 , 619 , 059 , 7 , 598 , 390 , 7 , 422 , 852 , 7 , 413 , 708 , 7 , 399 , 591 , 7 , 271 , 265 , 7 , 241 , 596 , 7 , 183 , 052 , 7 , 153 , 654 , 7 , 081 , 336 , 7 , 070 , 933 , 7 , 015 , 317 , 7 , 005 , 265 , 6 , 811 , 973 , 6 , 680 , 377 , 6 , 649 , 349 , 6 , 548 , 254 , 6 , 485 , 903 , 6 , 485 , 901 , each of which is expressly incorporated in its entirety . probes may be flourescent resonance energy transfer ( fret ) or non - fret type . see , u . s . pat . no . 6 , 150 , 097 , expressly incorporated herein by reference . c ) pcr is done with an annealing temperature of 60 ° c . for 50 cycles and quantified using a real time pcr machine . a range of 52 - 65 ° c . for the pcr would be acceptable . d ) values generated by demethylated probes are subtracted from values of methylated probes and a delta calculated . probe testing of logarithmic serial dilutions of synthetic hypomethylated and hypermethylated dna has shown a linear behavior ( r 2 = 0 . 98 ) of the delta between hypermethylated dna and hypomethylated dna ( delta = hypermethylated dna - hypomethylated dna ) over a wide range of dna dilution ( range is 4 log scale ) see fig2 a . log 10 transformation of demethylation index measures show a nonlinear fit ( r 2 = 0 . 9999 , df 2 ) see fig2 b . fig2 c shows the specificity of the assay . the probe detects demethylated dna at ˜ 180 folds in islet ( where β cells reside ) compared with liver and kidney which do not express insulin . in contrast , primers detect the demethylated dna at ˜ 80 fold . in other words probes used according to an embodiment of the present invention are 2 . 25 times more specific than primers the primers used in accordance with akirav et al . ( 2011 ). the present method extends the use of demethylated β cell derived dna as a biomarker of type 2 diabetes . the ability of the present assay to detect β cell loss in type 2 diabetes is clearly shown by the experimental results obtained with the use of the present method . fig3 a shows impaired glucose levels in patients with long - standing type 2 diabetes . fig3 b shows the increase in demethylated β cell dna ( i . e ., increase in methylation index ) in the serum of these patients , revealed as a significant difference ( p = 0 . 0286 ) from control by the use of the present probe technology . similar results are also observed in animal models of type 2 diabetes . fig3 c shows the use of primers from akirav et al . ( 2011 ) to analyze the same sample set , and failed to detect any significant difference ( p = 0 . 87 ) in methylation index between control and t2d patients . for pcr according to akirav et al ., ( 2011 ), shown in fig3 c was conducted for 40 cycles , with a melting temperature of 54 ° c ., using primers as follows : the second step real - time methylation - specific nested pcr according to akirav et al . ( 2011 ) was conducted with 50 cycles of amplification , and a melting temperature of 64 ° c ., with the following primers : fig4 a shows the ability of to detect elevated demethylated dna levels in the ob / ob leptin deficient mouse model of type 2 diabetes . these levels were correlated with elevated body weight , shown in fig4 b , and increased glucose levels , shown in fig4 c . although the present invention has been described in relation to particular embodiments thereof , many other variations and modifications and other uses will become apparent to those skilled in the art . it is preferred , therefore , that the present invention be limited not by the specific disclosure herein , but only by the appended claim . 1 . akirav e , kushner j a , herold k c ( 2008 ) β - cell mass and type 1 diabetes : going , going , gone ? diabetes 57 : 2883 - 2888 . 2 . bluestone j a , herold k , eisenbarth g ( 2010 ) genetics , pathogenesis and clinical interventions in type 1 diabetes . nature 464 : 1293 - 1300 . 3 . waldron - lynch f , herold k c ( 2009 ) advances in type 1 diabetes therapeutics : immunomodulation and β - cell salvage . endocrinol metab clin north am 38 : 303 - 317 . 4 . bougneres p f , et al . 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