Patent Application: US-201414771982-A

Abstract:
a method or assay for determining whether an individual is a hnf1a - mody carrier is described and comprising a step of assaying a biological sample from the individual to detect increased abundance of a micro rna molecule selected from mir103 , mir551b , or mir224 , or detect decreased abundance of a micro rna molecule selected from mir503 , and mir539 . increased abundance of one or more of mir103 , mir551b , or mir224 or decreased abundance of one or more of mir503 and mir539 , indicates that the individual is a hnf1a - mody carrier . a method for treating metabolic disorders , especially diabetes mellitus , is also described .

Description:
rat ins - 1 insulinoma cells inducibly expressing the human hnf1a - mody frameshift mutant pro291fsinsc - hnf1a under the control of a doxycycline - dependent transcriptional activator have been described previously [ 3 , 9 - 10 , 20 ]. the pro291fsinsc - hnf1a mutant has been shown to bind endogenous hnf1a , and to act as a dominant - negative transcription factor in vitro [ 4 ]. cells were cultured in rpmi 1640 at 6 mm glucose supplemented with 10 % fetal bovine serum ( fbs ) ( paa , cölbe , germany ), 2 mmol / 1 l - glutamine , 1 mmol / 1 pyruvate , penicillin ( 100 u / ml ), streptomycin ( 100 μg / ml ), 10 mmol / 1 hepes ( ph 7 . 4 ) and 50 μmol / l 2 - mercaptoethanol ( sigma , dublin , ireland ) [ 21 ]. measurement of hnf1a induction in ins - 1 cells was carried out using absolute qpcr . a hnf1a gene - specific pcr amplicon was prepared as a standard . the calibration curve was created by plotting the threshold cycle ( ct ) corresponding to each standard versus their corresponding log number of hnf1a standard ( expressed as cdna copy number of the hnf1a gene ). normalization was carried out using total rna values , as previously described [ 9 ]. database analysis was carried out to determine possible gene targets of the experimentally validated , differentially expressed mirnas . we employed the use of two different target prediction algorithms , miranda and targetscan , for our analysis . gene functional analysis was carried out using david to group putative gene targets into functional signalling kegg pathways . the number of genes ( count ) and the percentage of genes from the predicted lists are shown in table 1 . analysis of putative target genes for mir - 103 and mir - 503 revealed the insulin signalling pathway to be amongst the top signalling pathways targeted . analysis of putative target genes for mir - 103 also revealed ca 2 + signalling . analysis of putative target genes for mir - 224 revealed endocytosis and tgf - beta signalling . ins - 1 cells were cultured in t25 flasks in complete medium with or without 500 ng / ml doxycycline for 24 h or 48 h . cells were harvested for total rna using the qiagen mirneasy kit ( qiagen , hilden , germany ) according to manufacturer &# 39 ; s protocol . all downstream applications were performed on ice in an rnase free environment to prevent mirna degradation . thirty nanograms of total rna enriched in mirnas was converted to cdna using the taqman ® microrna reverse transcription kit ( roche - applied biosystems ), and preamplification was carried out on the cdna using megaplex preamp primers ( invitrogen ). taqman ® universal pcr master mix ( roche - applied biosystems ), was added to the samples which were then loaded onto taqman ® rodent microrna a + b cards set v2 . 0 ( invitrogen ) and run on a 7900ht system using sds software . each card contains 3 endogenous rat controls . the data was normalised to the endogenous control mammu6 ( mammu6 - 4395470 ). the controls u87 ( u87 - 4386735 ) and y1 ( y1 - 4386739 ) were normalised to the mannu6 . prior to calculating relative expression values , normalization was carried out by subtracting the average endogenous control sample ct from the individual mirna ct values . relative expression ( rq ) of mirna was calculated using the δδct method ; rq = 2 −( δδct ) . p - values were calculated using a t - test assuming two - tailed distribution in two samples of unequal variance . ins - 1 cells were cultured in 6 - well plates in complete medium with or without 500 ng / ml doxycycline for 24 h or 48 h . cells were harvested for rna enriched in mirnas using the qiagen mirneasy kit according to the manufacturer &# 39 ; s protocol . reverse transcription was carried out on 30 ng of rna using the taqman ® microrna reverse transcription kit , and taqman probes specific to the individual mirnas chosen ( mir - 103 , mir - 224 , and mir - 29a ). real - time pcr amplification was performed with the reverse transcription products using taqman ® 2 × universal pcr master mix without ung amperase , and mirna specific taqman probes ( roche - applied biosystems ): as mirna - 29a did not show any change of expression in the mirna array it was chosen as an internal control for the real - time pcr in ins - 1 cells overexpressing the pro291fsinc - hnf1a mutant . samples were run on an applied biosystems 7500 real - time pcr system with an initial denaturation at 95 ° c . for 10 min , followed by 60 cycles at 95 ° c . for 15 s and 60 ° c . for 1 min . potential gene targets of differentially expressed mirnas were predicted using the two public databases miranda ( http :// www . microrna . org / microrna / home . do ) and targetscan ( http :// www . targetscan . org /). the gene functional classification tool david ( http :// david . abcc . ncifcrf . gov / home . jsp ) was used to identify the signalling pathways to which the predicted gene targets belonged [ 22 - 23 ]. subjects with a clinical diagnosis of mody were recruited from the mody diabetes clinics in the mater misericordiae university hospital dublin in ireland . sequencing of the hnf1a gene was performed by integragen ( bonn , germany ) in 2006 - 2007 and the molecular genetics laboratory ( exeter , uk ) in 2008 - 2010 . genetically confirmed mody subjects included 31 cases with hnf1a mutations . the subjects with hnf1a mutations were from 11 pedigrees and the mutations included l17h ( n = 1 ), g207d ( n = 1 ), p291finsc ( n = 14 ), s352fsdelg ( n = 9 ), f426x ( n = 2 ), p379t ( n = 2 ), and ivs7 - 6g & gt ; a ( n = 2 ). the mutations named above have been previously published and are known to co - segregate in families with diabetes [ 24 - 25 ]. there were 10 normoglycaemic bmi - matched hnf1a - mody negative family members as well as which were available as a control group . all subjects underwent a clinical assessment including a full medical history and physical examination . details of the subjects &# 39 ; weight , height and blood pressure were recorded . a 75 g ogtt was performed on subjects after a 12 - h overnight fast with measurement of glucose , insulin and c - peptide at baseline and at 30 minute intervals for 120 minutes to determine the degree of glucose tolerance and insulin secretory response . in patients with diabetes , oral hypoglycemic agents were stopped at least 48 - h before the ogtt while , in those taking insulin , long - acting insulin therapy was stopped for 24 - h and short - acting insulin stopped for 12 - h prior to ogtt . the diagnostic criteria for the american diabetes association was used to define the degree of glucose tolerance . the oral glucose insulin sensitivity ( ogis ) was calculated as previously described [ 26 ]. the plasma glucose concentration was measured using beckman synchron dxc800 ( beckman instruments inc , brea , usa ). hba 1c was determined using high performance liquid chromatography ( menarini ha81 - 10 , rome , italy ). insulin and c - peptide were analyzed using immulite 2000 immunoassay ( siemens healthcare diagnostics , deerfield , ill ., usa ). the study was approved by the research ethics committee at the mater misericordiae university hospital dublin and all subjects gave informed written consent . total rna enriched with mirnas was isolated from hnf1a - mody carriers and mody - negative , non - diabetic family member sera using the total rna purification kit ( norgen , biotek cooporation , on , canada ). reverse transcription was carried out on 10 ng of rna using the taqman ® microrna reverse transcription kit , and taqman probes specific to the individual mirnas ( mir - 103 and mir - 224 ). individual mirnas from patient serum samples were detected by absolute qpcr . taqman assays for human mirnas hsa - mir - 103 and hsa - mir - 224 were obtained from applied biosystems . oligoribonucleotides corresponding to the mature sequence of each mirna were synthesized ( sigma aldrich ) and reverse transcribed to generate a standard curve . the oligoribonucleotides sequences used were : mir - 103 mature sequence : the standard curve was established using cdna generated from the 23 - bp or 25 - bp human mir - 103 / mir - 224 mature sequence amplicons , respectively . two microlitres of cdna were used as templates for pcr using the lightcycler 2 . 0 . the mir - 103 and mir - 224 amplicons were isolated and purified using qiaquick pcr purification kit ( qiagen ). the absolute quantity of the purified amplicons were measured by their absorbance at 260 nm and converted to the number of copies using the molecular weight of dna . the mir - 103 and mir - 224 amplicons were diluted to the concentration of 5 . 98 × 10 7 / 5 . 87 × 10 7 copies per μl ( stock solution ). when a standard curve was to be established for the real - time quantitative pcr assay , the stock solution was diluted over 7 orders of magnitude ( e . g . 5 . 98 × 10 7 , 5 . 89 × 10 6 , 5 . 89 × 10 5 , 5 . 89 × 10 4 , 5 . 89 × 10 3 , 5 . 89 × 10 , and 5 . 89 × 10 1 , copies per 2 μl ). all unknown sample concentration had to fall within this range . samples were run on the lightcycler 2 . 0 system ( roche ) with an initial denaturation at 95 ° c . for 10 min , followed by 70 cycles at 95 ° c . for 10 sec ( denaturation ) 60 ° c . for 20 sec ( annealing ) and 72 ° c . for 1 sec . the data were analysed using lightcycler software 4 . 0 ®. the normalized values ( dct ) from serum of hnf1a - mody carriers were compared with serum from mody - negative , non - diabetic family members . ins - 1 rat insulinoma derived cells were cultured in rpmi supplemented with foetal bovine serum ( 10 %) penicillin ( 100 units / ml ), streptomycin ( 100 μg / ml ), and glutamine ( 2 mm , sigma aldrich , dublin ). for experiments , cells were plated in 96 well plate ( corning , new york , usa ) at a density of 10 , 000 cells per well . after 24 h cells were transfected in optimem using dharmafect duo transfection reagent as per manufacturer &# 39 ; s instructions . either an antagomir targeting mir 224 or a control antagomir ( 100 nm , exiqon , vedbaek , denmark ) were cotransfected with plasmid encoding gfp ( 50 ng dna / well ). following transfection , cells were cultured for 72 h before fixation in paraformaldehyde ( 4 % for 15 min ). polyclonal anti - swine insulin antibody raised in guinea pig ( dakocytomation , stockport , uk ) was diluted 1 : 100 in h 2 o with 5 % goat serum and 0 . 1 % triton t100 ( sigma aldrich , dublin ) and 100 μl was added to each well for 1 h at room temperature . each well was then washed three times with 100 μl hank &# 39 ; s balanced salt solution ( sigma aldrich , dublin ) and anti - guinea pig igg ( h + l ) raised in goat with conjugated alexa fluor ® 568 ( diluted 1 : 100 in h 2 o with 5 % goat serum , invitrogen , dublin ) was added for 1 h at room temperature . live cells were stained with tmrm ( 20 nm ) or fixed and stained insulin antibody as described above . cells were then trypsinised and single - cell fluorescence intensity measured using a bd lsr ii flow cytometer . gfp was measured using a blue ( 488 nm ) laser with both 505lp and 525 / 50 nm emission filters and gfp positive cells were gated and taken as the transfected population . the yellow / green ( 561 nm ) was used for tmrm or alexa fluor ® 568 with 605 / 40 nm emission filter . data was exported as fcs files and analysis was carried out using cyflogic software . homeostatic blood glucose levels were measured in male and female animals at 3 , 6 and 10 week timepoints using bayer contour ® blood glucose meter . a small drop of blood (˜ 0 . 6 ul ) from the tail vein was collected and placed on a glucometer test strip . after 45 second developing time , the baseline blood glucose value is recorded ( mmol / l ) and the mouse returned to its cage . pancreatic insulin content as determined from 3 wk and 10 wk male animals . pancreata were removed and immediately frozen in liquid nitrogen , weighed and incubated o / n in acid - etoh ( 1 . 5 % hcl in 70 % ethanol ) at − 20 ° c . samples were then homogenised and incubated o / n at − 20 ° c . samples were centrifuged 15 min 2000 rpm at 4 ° c ., supernatant removed and neutralised with 1 : 1 volume tris ( 1m ph7 . 5 ). insulin content in acid - ethanol supernatant was determined with mouse insulin elisa ( mercodia ab ). statistical analysis was carried out in spss and data expressed as mean insulin concentration ug / mg pancreas ± sem . n = 3 per genotype . newborn male mice were used for the microrna analysis . mouse pancreases were harvested and immediately snap frozen in liquid nitrogen . rna enriched in mirnas was prepared from pancreas samples using the qiagen mirneasy kit ( qiagen , hilden , germany ). according to the manufacturers &# 39 ; protocol . for real - time quantitative pcr analysis 500 ng of total rna enriched in mirnas was reverse - transcripted using the taqman microrna reverse transcription kit ( life technologies , carlsbad , usa ) and individual taqman primer ( life technologies , carlsbad usa ): were used for pcr according to the manufacturer &# 39 ; s instructions . real - time pcr amplification was performed with the reverse transcription products using taqman 2 × universal pcr master mix without ung amperase , and mirna - specific taqman probes . the data was normalised to the endogenous control u6snrna ( assay id_001973 ). data were analyzed by the relative quantification ( δδct ) method . rat ins - 1 insulinoma cells overexpressing the gene encoding wild - type ( wt ) hnf - 1alpha or a dn mutant of hnf - 1alpha ( dn - hnf - 1alpha ) under control of a doxycycline - dependent transcriptional activator were cultured in rpmi 1640 at 11 . 1 mmol / l glucose supplemented with 10 % fetal bovine serum ( fbs_paa , cölbe , germany ), 2 mm l - glutamine , 1 mm sodium pyruvate , 100 u / ml penicillin , 100 μg / ml streptomycin , 10 mm hepes and 50 μm 2 - mercaptoethano ( asfari et al . 1992 ). ins - 1 cells were maintained at 37 ° c . in a humidified incubator gassed with 5 % co 2 . to generate the rno - mir224 expression plasmid which co - expresses egfp , the full length egfp sequence was subcloned from the pegfp - n1 ( clontech ) into the plvx - euro vector ( clontech ) using ecori and xbai restriction sites to generate plvx - egfp - puro . the rat mir - 224 pre - mir sequence was identified from the ensembl database and a 302 by sequence containing 110 bp 5 ′ to the pre - mir sequence was synthesised by gene synthesis ( eurofins ) and subcloned into the xbai site of the plvx - egfp vector to generate plvx - egfp - rno - mir - 224 . all constructs were verified by sequencing . one day before transfection , ins - 1 cells were dispersed with trypsin - edta solution . for transient transfection ins - 1 cells were grown in a 6 - well plate at the destiny of 2 × 10 5 cells per well . the 224plasmid ( 1 μg / well ) was incubated with 4 μl / well turbofect ( thermo fisher scientific , waltham , mass ., usa ) dilute in 195 μl optimem ( life technologies , carlsbad , calif ., usa ) for each 6 - well and incubated for 20 min at room temperature . while lipid / dna complexes were forming , the cell culture medium was removed and replaced with 800 μl optimem . dna / transfection reagent mix was then added to the cells . ins - 1 cells were exposed to the transfection cocktail for 4 h . the transfection cocktail was then removed and replaced with 2 ml normal cell culture medium or culture medium supplemented with 500 ng / ml doxycycline ( sigma , munich , germany ) for 48 h to induce the production of hnf - 1alpha . all assays were done 24 h or 48 h after transfection . to analyze the function of mir551b in ins - 1 cells we used a specific mir551b inhibitor , which is designed for specific in vitro silencing of mir551b . one day before transfection , ins - 1 cells were dispersed with trypsin - edta solution . for transient transfection ins - 1 cells were grown in a 6 - well plate at the destiny of 2 × 10 5 cells per well . cells were transfected with 75 nm of mircury lna has - mir551b inhibitor ( sequence 5 ′- 3 ′: exiqon , vedbaek , denmark ) or with a non - silencing negative control ( sequence 5 ′- 3 ′: exiqon , vedbaek , denmark ) using dharmafect duo transfection reagent ( thermo fisher scientific , waltham , mass ., usa ) according to the manufacturer &# 39 ; s instructions . briefly , 1 . 5 μl of mircury lna inhibitor ( stock solution 50 μm ) and 4 μl of transfection reagent were diluted with 194 . 5 μl opti - mem ( invitrogen carlsbad , calif ., usa ), and incubated for 20 min at room temperature , and then added to the cells . while lipid / dna complexes were forming , the cell culture medium was removed and replaced with 800 μl optimem . transfection mix was added to the cells and after 4 h the transfection cocktail was replaced with 2 ml normal cell culture medium or culture medium supplemented with 500 ng / ml doxycycline ( sigma , munich , germany ) for 48 h to induce the production of hnf - 1alpha . all assays were done 24 h or 48 h after transfection . to analyze the function of mir503 and mir539 in ins - 1 cells we used mirna mimics . this are small , chemically modified double - stranded rnas that mimic endogenous mirnas and enable mirna functional analysis by up - regulation of mirna activity . one day before transfection , ins - 1 cells were dispersed with trypsin - edta solution . for transient transfection ins - 1 cells were grown in a 6 - well plate at the destiny of 2 × 10 5 cells per well . cells were transfected with 50 nm of mirvana ™ mirna mimics for mmu - mir502 ( id : mc12867 ; mature mirna sequence or with mirvana ™ mirna mimic negative control # 1 , which has a unique sequence designed such that it does not target any human , mouse , or rat gene ( life technologies , carlsbad , calif ., usa ) using dharmafect duo transfection reagent ( thermo fisher scientific , waltham , mass ., usa ) according to the manufacturer &# 39 ; s instructions . briefly , 1 μl of mirvana ™ mirna mimics ( stock solution 50 μm ) and 4 μl of transfection reagent were diluted with 195 μl opti - mem ( invitrogen carlsbad , calif ., usa ), and incubated for 20 min at room temperature , and then added to the cells . while lipid / dna complexes were forming , the cell culture medium was removed and replaced with 800 μl optimem . transfection mix was added to the cells and after 4 h the transfection cocktail was replaced with 2 ml normal cell culture medium or culture medium supplemented with 500 ng / ml doxycycline ( sigma , munich , germany ) for 48 h to induce the production of hnf - 1alpha . all assays were done 24 h or 48 h after transfection . total rna was extracted using the mirneasy mini kit ( qiagen , hilden , germany ). 1 μg total rna was reverse - transcribed using 24 μg random hexamers ( thermo fisher scientific , waltham , mass ., usa ), 0 . 5 mm dntps , 5 × first - strand buffer , 0 . 01 mol / l dithiothreitol , and 200 units of superscript ii reverse transcriptase ( invitrogen carlsbad , calif ., usa ) in a final reaction volume of 20 μl . sybr green i - based real - time rt - pcr was performed on the taqman 7500 ( life technologies , carlsbad , calif ., usa ) using the quantitech sybr green pcr kit ( qiagen , hilden , germany ) as per manufacturers &# 39 ; protocol . the pcr was performed using the following thermal cycling protocol : 95 ° c . for 15 min , 95 ° c . for 15 sec following 58 ° c . for 35 s and 72 ° c . for 45 s for 40 cycles . in relative quantification the expression of insulin mrna was measured with respect to the reference gene β - actin , which was expressed constitutively and at the same level in all the samples analysed . the primers used to amplify insulin and β - actin ( purchased from sigma - aldrich , st louis , mo ., usa ) were : insulin for : results of mirna expression in cells were expressed as means ± sem . differences between treatments were analyzed by student &# 39 ; s t - test , as well as one - way analysis of variance ( anova ) and subsequent tukey &# 39 ; s tests . serum measurements and clinical data were given as median and inter quartile range ( iqr ) and compared by mann - whitney u - test and spearman correlation analysis . statistical analysis was conducted using spss ( ibm corp ., armonk , n . y .) and matlab ( the mathsworks , inc , natick , mass .). differences were considered to be significant at p & lt ; 0 . 05 . the invention is not limited to the embodiment hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention . shields b m , hicks s , shepherd m h , colclough k , hattersley a t , ellard s ( 2010 ): maturity - onset diabetes of the young ( mody ): how many cases are we missing ? diabetologia 53 : 2504 - 2508 . wang h , antinozzi p a , hagenfeldt k a , maechler p , wollheim c b ( 2000 ) molecular targets of a human hnf1 alpha mutation responsible for pancreatic beta - cell dysfunction . embo j 19 : 4257 - 4264 yamagata k , furuta h , oda n , et al . 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