Patent Application: US-81167301-A

Abstract:
this invention is directed to a dna sequence comprising a nucleotide sequence encoding a variant paraoxonase protein and to said variant paraoxonase protein as well as a method and a kit for detecting a risk of cancer , coronary or cerebrovascular disease , hypertension , type 2 diabetes , dementia , joint arthrosis , cataract , or sensitivity to organophosphorus compounds in a subject , the method comprising isolating genomic dna from said subject , determining the allelic pattern for the codon 102 of the paraoxonase encoding pon1 gene in the genomic dna , identification of ile101val mutation indicating said risk being increased and for targeting paraoxonase activity modulating therapies . further this invention relates to transgenic animals comprising a human dna molecule encoding said variant paraoxonase and to a method of phenotype - targeted gene sequencing .

Description:
in order to find new previously unknown functional mutations in the human pon1 gene , phenotype - targeted hierarchial sequencing was used . the serum paraoxonase activity was determined for over 1000 serum samples . dna samples of 10 persons with the lowest pon activity were first chosen for sequencing and they were sequenced through in all 9 exons with an abi prism 3100 genetic analyzer ( applied bio systems ). a new previously unknown human pon1 mutation was found in codon number 102 in exon number 4 , called pon ile102val , causing the change atc to gtc ; ile to val . after the new mutation was found , dna samples of 100 men with low paraoxonase activities were sequenced , and the mutation was present in 9 . 0 % of the subjects . finally 1 , 595 dna samples available in the kihd ( kuopio ischaemic heart disease risk factor study ) cohort were genotyped and the new mutation was found for 61 persons ; 3 . 8 % of the random population sample of men . a polymerase chain reaction was carried out as follows : the genomic dna was amplified in eight parts specific for the pon1 - gene and for its exons 1 to 9 . eight different amplifications were made , with eight different pcr primer pairs ( seq id no : 5 - 20 ); one pair for each exon except for the exons 2 and 3 which were amplified together . all 9 exons were sequenced . the kit or assay for use in the method according to the invention preferably contains the various components needed for carrying out the method packaged in separate containers and / or vials and including instructions for carrying out the method . thus , for example , some or all of the various reagents and other ingredients needed for carrying out the determination , such as buffers , primers , enzymes , control samples or standards etc can be packaged separately but provided for use in the same box . instructions for carrying out the method can be included inside the box , as a separate insert , or as a label on the box and / or on the separate vials . the method according to the invention for determining the allelic pattern of the codon in question is preferably carried out as a polymerase chain reaction , in accordance with known techniques . 3 the pcr primer pair for human paraoxonase ( pon 1 ) exon number 4 was as follow : 5 ′- ctcctccatggttataaggg - 3 ′ ( seq id no : 9 ) and 5 ′- cccagagtaagaacattattc - 3 ′( seq id no : 10 ) ( product size 315 bp ). the primers were designed by marja marchesani and they were delivered by the aiv institute , sequencing services ( kuopio , finland ). pcr amplification was conducted in a 25 μl volume containing 150 ng genomic dna ( extracted from peripheral blood ), 10 × pcr buffer , dntp ( 10 mm of each ), 20 pmol / μl of each primer , dna - polymerase ( 2u / μl ) ( dynazyme ™ dna polymerase kit , finnzymes , espoo , finland ). samples were amplified with a biometra uno programmable thermoblock ( biometra , göttingen , germany ) with pcr programme conditions as follows : 95 ° c . for 3 minutes , repeat following for 30 cycles : 95 ° c . for 30 seconds , 58 ° c . for 45 seconds , 72 ° c . for 45 seconds , 72 ° c . for 5 minutes , 4 ° c . hold . amplified pcr - products were purified using the qiaquik pcr purification kit ( qiagen , valencia , calif .). sequencing was made using a abi prism ® 3100 genetic analyzer ( applied biosystems , foster city , calif .). the abi prism ® 3100 genetic analyzer is a fluorescence - based dna analysis system of capillary electrophoresis with 16 capillaries operating in parallel , fully automated from sample loading to data analysis . the sequencing reactions were made by using the dna sequencing kit ; big dye ™ terminator cycle sequencing v . 2 . 0 ready reactions with amplitaq ® dna polymerase ( fs abi prism ®, pe biosystems , foster city , calif .). the sequencing primers were the same as the pcr primers : 5 ′- ctcctccatggttataaggg - 3 ′ ( seq id no : 9 ) or 5 ′- cccagagtaagaacattattc - 3 ′ ( seq id no : 10 ). cycle sequencing was made in the geneamp pcr system 9600 ( pe biosystems ) with the programme as follows : repeat the following for 25 cycles ; rapid thermal ramp to 96 ° c ., 96 ° c . for 10 seconds , rapid thermal ramp to 50 ° c ., 50 ° c . for 5 seconds , rapid thermal ramp to 60 ° c ., 60 ° c . for 4 minutes ( to perform cycle sequencing under standard conditions , abi prism ® 3100 genetic analyzer sequencing chemistry guide , applied biosystems ). dye terminator removal and sequencing reaction clean - up was made using multiscreen 96 - well filtration plates ( multiscreen ®- hv clear plates , millipore , bedford , mass .). after purification the samples were denaturated at 94 ° c . for 1 min and the sequencing was done using the abi prism ® 3100 genetic analyzer using microamp optical 96 - well reaction plates ( applied biosystems ). specifically genotyping was done by extracting dna from edta blood with a salting - out method after lysing red cells with 10 mm nacl / 10 mm edta . the 315 bp exon 4 pcr - product of the pon1 gene was digested with sau 3 ai restriction endonuclease ( new england biolabs , beverly , mass . ), mixed with 6 × loading dye solution and run in 2 . 0 % agarose gel electroforesis . identification of normal and mutant forms was based on different electrophoretic migration rates of the restriction fragments , resulting in distinct bands ( normal form ( ile102ile ); 196 bp , 100 bp , 19 bp , heterozygote form ( ile102val ); 215 bp , 196 bp , 100 bp , 19 bp and homozygote form ( val102val ); 215 bp , 100 bp ). serum paraoxonase activity was measured based on its capacity to hydrolyse paraoxon . 100 μl of diluted serum ( 25 - fold dilution in tris - hcl buffer , ph 8 . 0 ) was mixed with 100 μl of paraoxon ( paraoxon , dr . ehrensdorfer gmbh , augsburg , germany ) ( 0 . 1 g in 66 . 1 ml of tris - hcl buffer , ph 8 . 0 ). formation of p - nitrophenol was monitored photometrically at 405 nm ( at 30 c . ), as previously described . 12 testing for the risk of cancer , coronwy or cerebrovascular disease , type 2 diabetes or hypertension the study subjects were from the “ kuopio ischaemic heart disease risk factor study ” ( kihd ), a prospective population study to investigate risk factors for cardiovascular diseases , type 2 diabetes , hypertension , dementia and cancers . 3 - 17 , 19 , 20 the kihd study protocol was approved by the research ethics committee of the university of kuopio , finland . the study sample comprised men from eastern finland aged 42 , 48 , 54 or 60 years . a total of 2 , 682 men were examined during 1984 - 89 . all participants gave a written informed consent . a dna sample was available for 1595 men . all cancer cases in the health care have been reported to a national cancer registry in finland since 1953 . 18 our study cohort was record - linked to this cancer registry data by using the unique personal identification code ( social security number ) that all finns have . deaths in the cohort were obtained by record linkage to the national death certificate registry and hospitalizations by record linkage to the national hospital discharge registry . the history of hypertension and diabetes was assessed at baseline and at a 4 - year follow - up by self - administered questionnaire , checked by an interviewer . both at baseline and at the 4 - year follow - up examination , blood pressure and fasting blood glucose were measured using identical methods both at baseline and at the 4 - year follow - up . 16 , 20 the first occurrence of cancer after the kihd baseline examination was registered in the cancer registry during 1984 - 97 for 60 cohort members . the primary site was prostate for 15 cancers . there were 1246 men with no prior chd or cerebrovascular disease . of these , 342 were smokers and 904 non - smokers . of the smokers , 21 died of a cardiovascular cause by the end of 1998 . of the 515 men examined at baseline during 1984 - 86 , 36 developed an arthrosis ( icd - 10 m15 - m19 ) by the end of 1998 . of the 1107 non - smoking men , 23 developed a cataract ( icd - 10 h26 - h29 ) by the end of 1998 . the association of the pon1 ile102val genotype with the risk of hypertension and diabetes was studied among 1038 men who were re - examined 4 years after the baseline examination , see references 15 , 19 for details of the re - examination . for the analysis of the incidence of hypertension , hypertensive ( history of hypertension , antihypertensive medication or systolic bp 160 mmhg or more or diastolic bp 95 mmhg or more ) and obese ( body mass index 29 kg / m 2 or more ) men and those with a history of cancer were excluded , leaving 488 men for the analysis . for the analysis of the incidence of type 2 diabetes , men with a history of cancer or prevalent diabetes at baseline ( fasting blood glucose 6 . 7 mmol / l or more or treatment for diabetes ) were excluded , after which exclusion there were 967 men for the analysis . lipoproteins were separated from fresh serum samples using ultracentrifugation and precipitation . 13 , 14 cholesterol and triglyceride concentrations were measured enzymatically , plasma ascorbate and lipid - standardized plasma vitamin e concentration by hplc methods 16 , 20 serum ferritin and apolipoproteins with a ria 12 . the maximal oxygen uptake , a measure of cardiorespiratory capacity , was measured directly during a symptom limited exercise test . 15 information regarding medical history and medications was obtained by interview . smoking was recorded using a self - administered questionnaire and the dietary intake of nutrients was estimated by four - day food recording . 17 risk - factor adjusted relative risks of cancer , prostate cancer and cardiovascular death were estimated by multivariate cox proportional hazards modelling and those of incident hypertension and incident diabetes by multivariate logistic regression modelling . covariates were selected by forward step - up modelling , using p - value of 0 . 10 as entry criterium . missing values in covariates were replaced by grand means . tests of statistical significance were one - sided . the statistical analyses were performed with spss version 10 . 0 for windows . of all members of the study cohort , 61 ( 3 . 8 %) were val allele carriers of the pon1 gene ile102val polymorphism . to ascertain the penetrance of the pon1 102 mutation , serum pon activity was measured at the 11 - year re - examination for 783 cohort members as described above . the mean activity was 168 . 7 u / l in the wild ile — ile homozygotes vs . 70 . 7 u / l in 102val carriers ( p & lt ; 0 . 001 ). in a 2 - way analysis of variance ( n = 782 ), the ile102val polymorphism ( p & lt ; 0 . 001 ) was a stronger predictor of paraoxonase activity than the leu54met polymorphism ( p = 0 . 016 ). in a multivariate cox model adjusting for the strongest other risk factors in this cohort : maximal oxygen uptake , dietary vitamin c intake , smoking status ( current smoker vs . non - smoker ), body mass index , serum lipoprotein ( a ), dietary iron intake and apolipoprotein b , the relative risk of any cancer in the 102val carriers was 2 . 4 ( 90 % ci 1 . 0 to 5 . 5 , p = 0 . 052 ), compared with 102ile homozygotes ( p & lt ; 0 . 001 for the model , table 1 ). this association was stronger in 462 smokers with 24 incident cancers ( rr 3 . 2 , 90 % ci 0 . 9 - 10 . 8 , p = 0 . 060 ) than in 1107 nonsmokers with 36 incident cancers ( rr1 . 5 , 90 % ci 0 . 4 - 4 . 8 , p = 0 . 300 ). the risk of prostate cancer was 4 . 9 - fold ( 90 % ci 1 . 4 - 17 . 4 , p = 0 . 021 ) among 102val carriers compared with the wild homozygotes ( table 1 ). the model included maximal oxygen uptake , place of residence , serum hdl 2 cholesterol , histories of stroke and any atherosclerosis - related disease , cholesterol lowering medication , dietary iron intake and diastolic blood pressure as covariates . the risk of cataract was examined in non - smokers , because smoking is an overwhelmingly powerful risk factor for cataracts . among the 1107 non - smokers , the 102val carriers had a 3 . 8 - fold ( 90 % ci 1 . 1 - 13 . 0 , p = 0 . 038 ) risk of cataract in a cox model adjusting for blood glucose , blood leukocyte count , hair mercury content and the examination year 1989 ( table 1 ). smoking men who were pon1 102val carriers had a 4 . 9 - fold ( 90 % ci 1 . 3 - 18 . 1 , p = 0 . 023 ) risk of cardiovascular death , compared with the 102ile homozygotes ( table 1 ). the covariates included in the model were maximal oxygen uptake , history of any atherosclerosis - related disease , place of residence , serum apolipoprotein b level , plasma lipid - standardized vitamin e concentration ( protective ), examination year 1988 ( vs . any other ), and the serum fatty acid ratio ( saturated / sum of monoenes and polyenes ). among non - obese men , the pon1 102val carriers had a 2 . 9 - fold ( 90 % ci 1 . 3 - 6 . 5 , p = 0 . 019 ) risk of hypertension , compared with non - carriers ( table 2 ), when adjusting for serum triglycerides , chd in exercise test , dietary vitamin e intake ( protective ), frequency of hangovers , dietary retinol intake , and pon1 54 polymorphism . as arthrosis is a chronic , gradually developing disease , only men examined in the first three years ( 1984 - 6 ) were included in a logistic regression analysis ( table 2 ). the carriers of the 102val mutation had a 4 . 0 - fold ( 90 % ci 1 . 3 - 12 . 4 , p = 0 . 022 ) risk of developing an arthrosis during the follow - up , when adjusting for waist - to - hip circumference ratio , serum ferritin and dietary intakes of vitamin e and vitamin c . men with an 102val allele had a 3 . 2 - fold ( 90 % ci 1 . 1 - 9 . 3 , p = 0 . 039 ) risk of type 2 diabetes , as compared with 102ile homozygotes . covariates in the model were serum fatty acid ratio ( defined above ), serum ferritin concentration and family history of obesity . the mini mental state examination was used to assess the presence of cognitive impairment and the degree of dementia of the kihd participants aged 65 - 71 during 1998 - 2000 . the test examines orientation ( ten items ), registration ( three items ), attention and calculation ( five items ), recall ( three items ) and language ( nine items ). a correct response to each item scores 1 ( incorrect 0 ), which are summed to give a potential maximum score of 30 . higher scores indicate better cognitive function . the mean score was 25 . 5 ( sd 2 . 5 ) among the 26 carriers of the pon102 val allele and 26 . 4 ( sd 2 . 2 ) among 338 non - carriers for whom data were available ( one - sided p = 0 . 03 1 in t - test , exact p = 0 . 045 ). the mini mental state examination score was directly associated ( pearson &# 39 ; s correlation coefficient 0 . 14 , p = 0 . 008 , n = 359 ) with serum paraoxonase enzyme activity . this association remained statistically significant ( p = 0 . 012 ) after a statistical adjustment for age and socio - economic status , which were other strongest predictors of the score . 10 garin m c , james r w , dussoix p , et al . paraoxonase polymorphism met - leu54 is associated with modified serum concentrations of the enzyme . a possible link between the paraoxonase gene and increased risk of cardiovascular disease in diabetes . j clin invest 1997 ; 99 : 62 - 6 . 11 mackness b , mackness m i , arrol s , turkie w , durrington p n . effect of the molecular polymorphisms of human paraoxonase ( pon1 ) on the rate of hydrolysis of paraoxon . br j pharmacol 1997 ; 122 : 265 - 8 . 12 mackness m i , harty d , bhatnagar d , winocour p h , arrol s , ishola m , durrington p n . serum paraoxonase activity in hypercholesterolemia and insulin - dependent diabetes mellitus . atherosclerosis 1991 ; 86 : 193 - 9 . 13 salonen j t . is there a continuing need for longitudinal epidemiologic research ?— the kuopio ischaemic heart disease risk factor study . ann clin res 1988 ; 20 : 46 - 50 . 14 salonen j t , nyyssönen k , korpela h , tuomilehto j , seppänen r , salonen r . high stored iron levels are associated with excess risk of myocardial infarction in eastern finnish men . circulation 1992 ; 86 : 803 - 11 . 15 lakka t a , venäläinen j m , rauramaa r , salonen r , tuomilehto j , salonen j t . relation of leisure - time physical activity and cardiorespiratory fitness to the risk of acute myocardial infarction . n engl j med 1994 ; 330 : 1549 - 54 . 16 salonen j t , nyyssönen k , tuomainen t - p , mäenpää p h , korpela h , kaplan g a , lynch j , helmrich s p , salonen r . increased risk of non - insulin dependent diabetes mellitus at low plasma vitamin e concentrations : a four year follow - up study in men . brit med j 1995 ; 311 : 1124 - 7 . 17 ihanainen m , salonen r , seppänen r , salonen j t . nutrition data collection in the kuopio ischaemic heart disease risk factor study : nutrient intake of middle - aged eastern finnish men . nutr res 1989 ; 9 : 89 - 95 . 18 teppo l , pukkala e , lehtonen m . data quality and quality control of a population - based cancer registry . experience in finland . acta oncologica 1994 ; 33 : 365 - 9 . 19 everson s a , goldberg d e , kaplan g a , et al . hopelessness and risk of mortality and incidence of myocardial infarction and cancer . psychosom med 1996 ; 58 : 113 - 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