Patent Application: US-15145305-A

Abstract:
the invention relates to methods for diagnosing a person &# 39 ; s susceptibility for having an increased risk for the development of atherosclerosis and a diabetic person &# 39 ; s susceptibility for having an increased risk for the development of diabetic retinopathy . the invention relates further to methods for treating persons diagnosed for having increased risk for the development of said diseases , in order to prevent the development of said diseases . the invention also concerns methods to investigate or screen pharmaceuticals or genetic aims useful in the treatment of said diseases , by using an animal model including a transgenic animal .

Description:
neuropeptide y ( npy ) is a 36 - amino - acid neurotransmitter widely present in the central and peripheral nervous systems . npy has multiple actions , which control body energy balance and cardiovascular function . we have recently demonstrated that the subjects having pro7 in the signal peptide of npy have higher serum cholesterol and apolipoprotein b levels when compared to individuals having wildtype ( leu7 / leu7 ) signal peptide sequence . the present invention is based on a study of the association of leu7 to pro polymorphism of the npy gene with common carotid intima - media - thickness ( imt ) assessed by ultrasonography cross - sectionally from the 10 - year follow - up study of newly diagnosed patients with type 2 diabetes ( 81 patients , 41 males , mean age 67 . 1 years ) and in non - diabetic subjects ( 105 subjects , 48 males , mean age 65 . 5 years ) who were genotyped for leu7pro polymorphism in prepronpy gene . the carrier frequency of the pro7 substitution was 9 . 9 % in diabetic patients and 14 . 3 % in control subjects ( p = 0 . 360 ). the mean common carotid imt was in non - diabetic subjects without leu7pro polymorphism 1 . 04 ± 0 . 02 and with it 1 . 14 ± 0 . 04 mm ( p = 0 . 156 ) and in diabetic patients 1 . 18 ± 0 . 03 and 1 . 58 ± 0 . 21 mm ( p = 0 . 004 ), respectively . in the analysis of covariance of the entire group the mean common carotid imt was independently associated with the leu7pro - polymorphism ( f = 5 . 165 , p = 0 . 024 ). the model included age , gender , diabetes , clinical macrovascular disease , smoking , systolic blood pressure and ldl - cholesterol . furthermore , diabetic patients having the pro7 in prepronpy had significantly more often diabetic retinopathy ( p = 0 . 04 ) when compared to patients with the leu7 / leu7 genotype . the present study indicates that the presence of pro7 substitution in the prepronpy is strongly associated with increased carotid atherosclerosis in diabetic and non - diabetic subjects , even after adjustment for known risk factors . furthermore , this is the first evidence that pro7 in the prepronpy increases the risk of type 2 diabetic patients to develop diabetic retinopathy . the dna sequence or the mutant signal peptide or said peptide associated with any other cleavage product of prepronpy can be used for screening a subject to determine if said subject is a carrier of a mutant npy gene . the determination can be carried out either as a dna analyse according to well known methods , which include direct dna sequencing of the normal and mutated npy gene , allele specific amplification using the polymerase chain reaction ( pcr ) enabling detection of either normal or mutated npy sequence , or by indirect detection of the normal or mutated npy gene by various molecular biology methods including e . g . pcr - single stranded conformation polymorphism ( sscp )- method or denaturing gradient gel electrophoresis ( dgge ). determination of the normal or mutated npy gene can also be done by using restriction fragment length polymorphism ( rflp )- method , which is particularly suitable for genotyping large number of samples . the determination can also be carried out at the level of rna by analysing rna expressed at tissue level using various methods . allele spesific probes can be designed for hybridization . hybridization can be done e . g . using northern blot , rnase protection assay or in situ hybridization methods . rna derived from the normal or mutated npy gene can also be analysed by converting tissue rna first to cdna and thereafter amplifying cdna by an allele spefic pcr - method and carrying out the analysis as for genomic dna as mentioned above . alternatively , the determination can be carried out as an immunoassay where a sample is contacted with an antibody capable of binding the signal peptide or said peptide associated with any other cleavage product of prepronpy . antibodies can be raised against normal or mutated prepronpy or more specifically against normal or mutated signal peptide part of the npy . the production of antibodies can be done in experimental animals in vivo to obtain polyclonal antibodies or in vitro using cell lines to obtain monoclonal antibodies . a person diagnosed for having an increased risk for the development of atherosclerosis , or a diabetic person , diagnosed for having an increased risk for the development of diabetic retinopathy , can be treated for the prevention of developing any of said diseases administering to said subject an effective amount of an agent counteracting the influence of the mutated npy gene . this can be done by specific gene therapy aimed to repair the mutated npy sequence , or by administering pharmacotherapies , which are aimed to modulate synthesis , release or metabolism of the endogenous npy , or to interact in a specific manner at npy target sites by modulating effects of npy with specific npy receptor proteins . currently , five different subtypes of npy receptors have been cloned and characterized ( y1 - y5 receptors ) and drug molecules specifically interacting with these npy receptors have been synthesized . the pharmacotherapy described is not limited to only these named receptors or mechanisms , but also covers other npy receptors and related mechanisms to be discovered including the secretion of npy . counteracting the influence of the mutated npy gene in a patient by using an antisense therapy or gene switching or replacement , which includes targeted correction of disease - related mutation or site - directed inactivation of the mutant allele by homologous recombination . the antisense therapy refers to methods designed to impair translation through direct interactions with target messenger rna ( mrna ). this can be accomplished by applying a targeted oligonucleotide , which forms watson - crick base pairs with the messenger rna whose function is to be disrupted . the inhibition of gene expression by antisense oligonucleotide depends on the ability of an antisense oligonucleotide to bind a complementary mrna sequence and prevent the translation of the mrna . it is possible to correct a single mutant base in a gene by using an oligonucleotide based strategy ( giles et al ., 1995 ; schwab et al ., 1994 ; yoon et al ., 1996 ). a short , 7 or 8 bases , oligonucleotide is enough to posses an antisense activity and specificity , which depends greatly on the flanking sequences of the target rna . binding should be enough to promote stable binding and rnase h - mediated cleavage . we are counteracting the influence of the mutated npy gene by using a short , allele specific oligonucleotide , which includes the sequence of mutated part : . . . cga ct / cg ggg . . . ( mutated base marked on bold ) ( seq id no : 8 ). this can be accomplished by using oligonucleotides of various lengths , but all recognizing the mutated base sequence . according to the predicted secondary structure of the prepronpy mrnas ( fig2 and 3 ), the best target sequence is between − 9 and + 2 bases around the mutation i . e ., a sequence targeting to 5 ′- ac aag cga ccg g - 3 ′ ( seq id no : 9 ). this sequence contains ‘ bulbs ’ which are known to enhance the binding of oligonucleotide to the target mrna . it is possible to use unmodified oligonucleotides , but to increase their stability , nuclease resistance , and penetration to the nucleus , several modifications of oligonucleotide can be used . a relatively large number of modified pyrimidines have been synthesized , mainly c - 2 , c - 4 , c - 5 , and c - 6 sites , and incorporated into nucleotides . also purine analogs can be synthesized and incorporated into oligonucleotides . the 2 ′ position of the sugar moiety , pentofuranose ring , is substituted with methoxy , propoxy , o - alkoxy or methoxyethoxy groups . a new backbone for oligonucleotides that replace the phosphate or the sugar - phosphate unit has been made , like c - 5 propynylpyrimidine - modified phosphothioate oligonucleotides . also chimeric oligonucleotides with 5 ′- and 3 ′- ends are modified with internucleotide linkages , like methylphosphorothioate , phosphodiester , or methylphosphonate can be used . a relatively new technique is conformationally restricted lna ( locked nucleic acid ) oligonucleotides and peptide nucleic acids . bioengineered ribozymes are structurally different , but their specificity also relay on the recognition of the targeted mrna sequence . gene replacement or gene switching techniques inactivate the mutated gene sequence and introduce a corrected one . this can be accomplished by transfecting exogenous gene with normal coding sequence and blocking mutant coding sequence with antisense oligonucleotide . also a technique with only introducing a corrected normal sequence without disrupting the mutated sequence could be use . this could be used in heterozygous cells i . e . cell carrying one normal allele and one mutated allele resulting in an overexpression of normal alleles . also homozygous mutant cells could be treated resulting in a dominant positive - effect i . e . the normal allele is expressed in higher degree than the mutant allele . influence of the mutated npy sequence on the function of npy gene can be investigated in transgenic animals . a transgenic animal can be generated using targeted homologous recombination methodology . both normal and mutated sequence of human npy signal peptide ( or any dna sequence comprising a nucleotide sequence encoding a prepro - neuropeptide y ( prepronpy ) or part thereof encoding the amino acid sequence of the mature mouse or human mature npy peptide , where either i ) the leucine amino acid in position 7 of the signal peptide part of said prepronpy has been replaced by proline or ii ) the leucine amino acid in position 7 of the signal peptide part of said prepronpy is unchanged ) will be introduced into the sequence of npy gene to replace the endogenous signal peptide sequence . under these conditions , the endogenous npy gene functions otherwise normally , but the synthesis of the prepronpy is regulated by either normal or mutated human npy signal peptide sequence . this transgenic model can be used to investigate in a very specific manner the physiological importance of the mutated npy gene . it also will provide an ideal preclinical model to investigate and screen new drug molecules , which are designed to modify the influence of the mutated npy gene . this study was a cross - sectional analysis from the 10 - year examination of a cohort of patients with type 2 diabetes and nondiabetic control subjects followed up from the time of diagnosis , as described earlier in detail ( 16 - 22 ). in brief , the original study comprised 133 patients with newly diagnosed type 2 diabetes , aged 45 to 64 years , and 144 nondiabetic control subjects randomly selected from the population register . the baseline study was carried out during the years 1979 - 81 and all subjects were collected from a defined area in eastern finland ( 16 ). all the subjects were invited for the 5 - and 10 - year follow - up examinations during the years 1985 - 86 ( 17 ) and 1991 - 92 ( 18 - 19 ), respectively . during the 10 - year follow - up 36 ( 27 %) diabetic patients and eight ( 6 %) nondiabetic subjects died , mainly due to cardiovascular diseases ( 18 ). at the 10 - year examination , carotid ultrasonographic examinations ( 20 - 21 ) were performed for 84 ( 63 %) of the original diabetic and 119 ( 83 %) of the nondiabetic populations and genotype analysis was made for all these except for three diabetic and one non - diabetic subject . the study was approved by the ethics committee of the university of kuopio . the assessment of medical history and cardiovascular diseases , the use of medication , smoking , blood pressure , body - mass index ( bmi ) and waist - to - hip circumference ratio have been described in detail previously ( 18 - 22 ). the group “ macrovascular disease ” refers to subjects with any previously defined evidence of myocardial infarction , stroke or intermittent claudication . an oral glucose tolerance test was performed by using a glucose dose of 75 g . the impaired glucose tolerance in control subjects was classified according to the who criteria ( 23 ). the collection of blood specimens and the measurement of serum lipid and lipoproteins by ultracentrifugation and precipitation methods , apolipoprotein b , plasma glucose and plasma insulin have been likewise presented previously ( 19 - 22 ). prepronpy genotype was determined by restriction fragment length polymorphism ( rflp ) analysis from dna extracted from the subjects peripheral blood by an investigator unaware of phenotype . briefly , the polymorphism appears as a thymidine ( 1128 ) to cytosine ( 1128 ) substitution generating a bsi ei restriction site , which was used to genotype the subjects for the leu7pro polymorphism , as described previously ( 15 ). the pcr products were digested by bsi ei ( new england biolabs , inc . beverly , mass ., usa ) and digestions were analyzed by electrophoresis on 2 % agarose gel . the high - resolution b - mode ultrasonographic imaging protocol was designed to ensure the valid and reliable identification of arterial carotid references and the definition of near - wall and far - wall interfaces , as described previously in more detail ( 20 - 21 , 24 ). briefly , the carotid artery was divided into two segments on the basis of arterial anatomy and geometry . the key anatomic features defining these segments were the proximal origin of the bulb ( carotid bifurcation ) and the tip of the flow divider , which separates internal from external carotid arteries . in longitudinal arterial images , the adventitia - media and the intima - lumen interfaces on the far wall were the specific anatomic bondaries defining the imt . two certified sonographers performed the carotid ultrasound examinations . a biosound phase two ultrasound device equipped with a 10 - mhz annular array probe was used . video - recorded examinations were quantitatively analyzed at a central laboratory using a computer - assisted reading procedure ( 24 - 25 ). the mean maximum of the far wall bilaterally was used as the measurement of the common carotid imt . our a priori hypothesis was that the subjects having pro7 substitution in prepronpy have higher mean imt compared to the subjects having wild type prepronpy ( leu7 / leu7 ). associations of leu7pro polymorphism with continuous variables were calculated using student &# 39 ; s t - test and for categorized variables by chi square test . the association of common carotid imt with leu7pro polymorphism was further analyzed by analysis of covariance ( ancova ) controlling for the effects of selected covariates . variables with skewed distribution ( eg . carotid imt , insulin ) were analyzed after logarithmic transformation . p - value equal or less than 0 . 05 was considered statistically significant . all statistical analyses were conducted with procedures from spss - unix . the frequency of c1128 allele frequencies was not significantly different between non - diabetic ( 14 . 3 %) and diabetic ( 9 . 9 %, p = 0 . 36 ) groups . the characteristics of non - diabetic and diabetic subjects for leu7 / leu7 and pro7 /- groups are presented in tables 1a - b . no differences in age , gender , body mass index , waist - to - hip - ratios , blood pressure levels and the frequencies of macrovascular disease were found between the genotype groups within the non - diabetic and diabetic groups . ldl - cholesterol was higher in non - diabetic subjects with leu7 / pro - polymorphism than in those without ( p = 0 . 05 ), as we have reported previously ( 15 ). although apolipoprotein b levels tended to be higher in pro7 /- group than in leu7 / leu7 - group , the differences were not statistically significant . our previous study included only lean subjects without any medication known to affect cholesterol metabolism ( like beta - blockers or diuretics ) of the present non - diabetic group ( 15 ). in other lipoproteins no evident differences were found , and interestingly , in diabetic patients there was no association with serum cholesterol , even when subjects were analyzed according to median body mass index ( data not shown ). the mean common carotid imt was about 25 % higher in diabetic patients with pro7 allele than in those without it ( p = 0 . 004 ) and the respective increase in imt was 9 % in non - diabetic subjects ( p = 0 . 156 ). in the analysis of covariance both groups combined ( table 2 ) the independent predictors of common carotid imt were age , pro7 allele , diabetes , systolic blood pressure , and macrovascular disease . furthermore , those diabetic patients having the pro7 substituion in the prepronpy had significantly accelerated rate of diabetic retinopathy ( p = 0 . 04 ), when compared to diabetics with the leu7 / leu7 - genotype . our findings based on elderly finnish non - diabetic and diabetic subjects indicates that the pro7 allele of prepronpy is strongly associated with increased carotid atherosclerosis , and even more markedly in diabetic patients . this finding is of importance , because an increase in the thickness of imt of carotid arteries increases the risk for cardiovascular events in a linear fashion even before clinical manifestations of cardiovascular diseases ( 26 ). in addition , the presence of pro7 polymorphism in the prepronpy was significantly associarted with the rate of diabetic retinopathy . the pro7 allele was also associated with high serum ldl cholesterol levels and apolipoprotein b - levels in lean non - diabetic subjects ( 15 ), but this was not found in diabetic patients regardless of their body weight . type 2 diabetes is a state characterized by markedly increased risk of atherosclerosis and although known risk factors contribute largely to the occurrence of diabetic macrovascular diseases ( 27 ), a large proportion of this vascular burden remains unexplained and search for other potential environmental , metabolic and genetic contributors are warranted . in this study we show for the first time that diabetic patients with pro7 allele have higher carotid imt than those with leu7 / leu7 - genotype . although this finding was based on a limited number of subjects , the lack of association of pro7 allele with other risk factors measured in diabetic patients makes the finding more intriguing . as non - diabetic control group included subjects with impaired glucose tolerance as any population - based study does and therefore , glucose tolerance is in a way continuum in this study population , we combined the groups in order to increase the statistical power of the study for the analysis of covariance . in this analysis age , diabetes , systolic blood pressure and clinical macrovascular disease were , as previously reported ( 21 ), powerful explanatory variables of carotid imt . interestingly , the effect of npy genotype remained statistically significant in this analysis . other cardiovascular risk factors except fasting insulin in non - diabetic subjects were not associated with npy genotype in either group . the selective mortality may cause bias in the interpretation , as in any cross - sectional analysis . however , as ldl - cholesterol - levels were constantly higher during the whole 10 - year follow - up in lean non - diabetic control subjects with pro7 allele and , on the other hand , the genotype effect on carotid imt was more marked in diabetic patients who had high cardiovascular mortality from the time of diagnosis ( 18 ), it is likely that this study under - estimates this association . why could then npy enhance the development of atherosclerosis ? first , this effect may be mediated by the effects of the prepronpy genotype on ldl - cholesterol metabolism ( 15 ). however , this effect is modulated by body weight ( 15 ) and as judged from the present study , no effect was seen in type 2 diabetic patients in this regard ( more detailed arialysis of lipoproteins assessed either cross - sectionally or longitudinally gave no further insights in this regard ). second , npy may have angiogenic properties that could be implicated in the development of atherosclerosis . npy has been shown to act as a smooth muscle mitogen ( 28 ), to stimulate attachment , migration , dna synthesis ( 29 ), and the formation of capillary tubes by human endothelial cells ( 4 ). minor proportion of circulating npy level is derived from endothelial cells and this endothelially derived npy may act as an autocrine angiogenic factor even at very low concentrations ( 4 ). subjects with pro7 substitution in prepronpy may therefore be predisposed to increased arterial wall thickening seen as increased intima - media thickening of carotid arteries , because of impaired function of endothelial npy . third , npy is an important modulator of autonomic nervous system . majority of circulating npy is derived from the perivascular sympathetic nerve endings , and the level of npy is correlated to those of norepinephrine ( 30 ). autonomic nervous dysfunction is an independent predictor of cardiovascular mortality in patients with type 2 diabetes , as demonstrated from this study population ( 22 ). the mechanisms behind cardiovascular diseases and autonomic nervous dysfunction are speculative , but our unpublished observations suggest that cardiac autonomic regulation is altered in subjects with those with pro7 substitution in the prepronpy . therefore , we suggest that atherosclerosis may be associated with gene ( s ) involved in vascular development , lipid metabolism and autonomic nervous function and the recently found gene variant ( 15 ) in npy is the first one in this respect shown to be related to accelerated atherosclerosis . in conclusion , these results indicate that the presence of pro7 substitution in the prepronpy is associated with ultrasonographically assessed carotid atherosclerosis in finnish diabetic and non - diabetic subjects . furthermore , this study provides first evidence that the pro7 in the prepronpy is also associated with increased rate of diabetic retinopathy in niddm ( type 2 diabetes ) patients , which could be potential target for drug development . it will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . table 1a the clinical characteristics of the study population according to leu7 / pro - genotype in nondiabetic subjects . leu7 / leu7 pro7 /- characteristic n = 90 n = 15 p - value age ( years ) 65 . 5 ± 0 . 6 65 . 5 ± 1 . 1 0 . 982 male gender ( n , %) 43 ( 48 ) 5 ( 33 ) 0 . 298 body mass index ( kg / m 2 ) 27 . 8 ± 0 . 5 28 . 4 ± 1 . 2 0 . 611 macrovascular disease ( n , %) 11 ( 12 ) 4 ( 27 ) 0 . 139 smoking history ( n , %) 23 ( 26 ) 5 ( 33 ) 0 . 528 treatment for hypertension 26 ( 29 ) 6 ( 40 ) 0 . 387 ( n , percentage ) systolic blood pressure ( mmhg ) 149 ± 2 148 ± 3 0 . 863 diastolic blood pressure ( mmhg ) 85 ± 1 . 1 85 ± 3 0 . 847 fasting serum insulin ( mu / l ) 11 . 0 ± 0 . 6 14 . 8 ± 2 . 2 0 . 056 impaired gluclose tolerance ( n , %) 11 ( 12 ) 1 ( 7 ) 0 . 531 mean of common carotid imt 1 . 04 ± 0 . 2 1 . 14 ± 0 . 4 0 . 156 ( mm ) serum apolipoprotein b ( mg / l ) 1 . 04 ± 0 . 03 1 . 12 ± 0 . 08 0 . 285 serum hdl cholesterol ( mmol / l ) 1 . 34 ± 0 . 03 1 . 27 ± 0 . 08 0 . 403 serum ldl cholesterol ( mmol / l ) 4 . 11 ± 0 . 09 4 . 61 ± 0 . 30 0 . 05 serum total cholesterol ( mmol / l ) 6 . 29 ± 0 . 11 6 . 72 ± 0 . 37 0 . 153 serum triglycerides ( mmol / l ) 1 . 81 ± 0 . 12 1 . 65 ± 0 . 18 0 . 811 waste - 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