TWI357442B - Risk prediction for hypertension, elevated plasma - Google Patents

Description

1357442 IX. Description of the invention: [Technical field to which the invention pertains] * The present invention relates to a high risk of hypertension, a high triglyceride and a genetic risk factor of metabolic syndrome and related components and applications thereof. [Prior Art] Asking blood pressure is the main risk factor for cardiovascular disease, and it is a serious global public health. Nearly one billion people in the world suffer from this disease (Mein CA, 2^04) ° Evidence from family and epidemiological studies indicates that hypertension is derived from genetic and cyclic factors, complex interactions (HaiTap SB , Chapter24 in ref) 2. Twin studies and separation analysis showed that 2〇%-40% of blood pressure (BP) changes were attributed to genetic factors (Wai^R, 1990)3. For several Mendelian hypertension traits and hypertension experimental models, the genetic localization of sputum blood pressure genes has made significant progress (MeinCA, 2〇〇4). However, the common genetic variation of this human complex trait has not yet been elucidated (Mein CA, 2〇〇4) ι. In studies of patients with familial mixed hyperlipidemia (FCHL) and familial triglyceride (FHTG), hypertension is more common in the population than in the normal population (Hopkins PN) Et al., 2003) 4. In addition, triglycerides (TG) are associated with blood pressure, and hypertriglyceridemia in hypertensive patients usually exceeds expectations. These clustering phenomena (clusteringphen〇men〇n) have recently been considered as part of the metabolic syndrome (MS) and are characterized by a group of diseases, including high body mass index (BMI) or high waist circumference and high triglycerides. High-density lipoprotein cholesterol (HDL-C), high blood pressure, and glucose intolerance (GM, #公极j 1996)8. A variety of evidence has shown that metabolic syndrome plays an important role in the development of cardiovascular disease (Grundy SM et al, 2004). Lipoprotein lipolytic enzyme (LPL) is a key leaven that participates in the metabolism of "TG-rich particles" into free fatty acids and releases them into the circulation.

1357442 I. Kidney Bean has been shown to have a heterozygous activity-deficient heterozygote that tends to have a southern concentration of fasting TG and hypertension compared to wild-type (Sprecher DL et al, 1996) i〇 (William S RR et al., 1994)11. In addition, LPL activity abnormalities are associated with insulin resistance, obesity, diabetic dyslipidemia, chyl〇micr〇naemia, atherosclerosis, and angina pectoris (Mead Paw Exfoliation 2〇〇2) 12 ( Goodarzi MO, 2004) 13 (Kastelein John JP et al, 2000) m. Therefore, we have included ZPZ as one of many candidates in the Hypertension Gene Location Program. In our report (Pan et al., 2000), using the affected sib-pair method, we found markers in the early-onset hypertension population of Chinese Han Chinese in Taiwan (D8S1145). , p = 0.0284) There is a positive signal. The association of quantitative traits of systolic blood pressure with genetic markers near LPL is also documented in the diabetes family in Taiwan (Wu, et al., 1996) i6. Although the association between genotype and blood pressure has been controversial in Caucasian studies (Hunt et al, 1999; Clee SM, 2001) 17'18, recent independent studies have shown that Chinese Han nationality is more versatile and The positive correlation and association of hypertension, and further support the role of the role in the etiology of hypertension (Yang WJ, 2003; Ma YQ, 2002)19,20. Therefore, it is necessary to search for risk predictor gene markers and treatments that are effective for the above conditions. SUMMARY OF THE INVENTION In order to locate a single set of genes for hypertension and related diseases and to understand the effects of genetic variation on various aspects of this complex trait, we mapped the resolution of the LPZ region for early-onset hypertension, and the results showed two common The Yufan single genotype is associated with hypertension, high triglycerides, and/or metabolic syndrome (MS). The first single set of genes in the soil (C/AA/T single set of genes) contains two single nucleotide polymorphisms (snps) in the intron 3 to intron 4 region of the 丄fan gene: rs343(9) CBI gene pool NT-030737, position 7621708), rs252 (NC762 gene bank, NT-030737, position 7622153) and rs253 (1357442 in the NCBI gene bank, position 7622338 of ΝΤ_030737). For this risk single set of genes, the nucleotide acids in these three positions are C, AA and T, respectively. It has a significant correlation with diamylglycerol (TG) south (p = 0.005) and high gold pressure combined triglyceride (TG) bias ifj (p < 0.0001). The second single set of genes (GTAG single set of genes) contains four SNPs in the intron 5 to intron 6 region of the gene: AF050163-2500 (position 7624101 of NT_030737 in the NCBI gene bank), rs269 (NCBI gene) In the library, the number 7624588 of NT_030737), rs270 (the position of 7624597 of NT-030737 in the NCBI gene bank) and rs271 (the position of 7624462 of B NT_030737 in the NCBI gene bank). A single set of genotypes that would increase risk, the nucleotides at these four positions are G, T, A, and G, respectively. This single genotype of GTAG has a significant correlation with hypertensive combined triglyceride (TG) high disease (ρ<〇.〇〇〇ι). In addition, 'the person carrying the above two risk single sets of genes is suffering from high blood pressure (丨5), high triglyceride (1.9), hypertension combined with high triglyceride (23) and metabolic syndrome (1.8). 'The highest and most obvious hazard comparison value.

Therefore, the present invention provides a use of grasping as a target for hypertension, triglyceride (four), sciatic syndrome, and other lake diseases. The capture gene sequence or protein sequence can be used to evaluate = two. High levels of diglyceride, metabolic syndrome and other related diseases and medical conditions. Especially across introns 3 to introns 4 or = = use, such as C/mine* single set = t in addition to value genes, others with any high blood waste, archabolic syndrome or other dysentery money medical illness Said '〗 〖, marked the butterfly cap. Therefore, these other soil factors are used to assess the risk of these diseases and symptoms. 1 β inheritance “) The present invention may also provide milk as a target for the treatment, measurement and development of high ♦ triglyceride, 7 1357442 I metabolic syndrome and related diseases and symptoms. For example, Variants can be used in in vitro and in vivo assays to screen for drugs that bind to lpz and its variants, inhibit LPL activity, or restore LPL activity. Drugs with • can be analyzed step by step to determine Treatment or prevention of high blood pressure, high levels of triglycerides, metabolic syndrome and related diseases and medical symptoms. Particularly suitable for LpL containing C/AA/T and/or GTAG single genotypes. * The present invention also provides a nucleic acid comprising a mutation or a polymorphism, which is associated with hypertension, φ diglyceride, metabolic syndrome or a related disease or medical condition. The present invention also provides mutation or polymorphism. Proteins or peptides, in particular, can provide antibodies that recognize variants of two proteins or peptides, but non-wild-type lipoprotein lipolytic enzyme proteins. Containing nuclei, and complementary thieves and host cells are also provided. In particular, they can be used for debt. /Bei J and four blood pressure, high diglyceride, metabolic syndrome or related to the mutation or polymorphism (four) or probe is also provided. The 疋 contains any of the above and the instructions for use [Embodiment] The sister gene is associated with hypertension, triglyceride secret and metabolic syndrome. It is located in the 3 to intron 4 region - single set of base chat / aa / t single set of genes} and _ upper 6 regions A single set of genes (gtag single set of genes) can predict the individual risk of a "single 4" disease. In addition, other markers that are balanced with any single set of genes also have __. The present invention can be turned over to Asian ethnic groups, S Gasolians, cautious persons, and others. For other applications, unless otherwise noted, the details of the details of the present invention are defined as follows. 1357442 Defining LPL with blood pressure and trisodium citrate High and stagnation syndrome related to death and triglyceride is the main component of two metabolic symptoms, and may be ^^^^(5) This 'speak'--the main lipid metabolism gene' was selected as an early-onset face One of the candidates for the (10)TM) family of sputum gray-pressed genes. Strictly raised 59 cores The ΥΗΤΝ family, with 25 pairs of brothers and sisters to her (10) _ initial chain analysis study, followed by 345 ethnic 384 parental trios (trios) stub-related confirmation experiments. We tested the milk scorpion by the following steps Relationship between blood pressure and related phenotypes: microsatellite markers, linkage disequilibrium analysis, single genotype construction, and correlation analysis (Example 1_5). (10) Microsatellite markers and YHTN in genes Coupling. Unbalanced transmission test is not located in the inclusion = 3_4 # C / A / T single set of base _ with triglyceride (four) is high (P = 0.00? and high blood pressure combined with triglyceride high two diseases (four) The correlation between the single genotype GTAG and the high sputum and triglyceride high phenotype (pO.O00l) was significantly correlated. In addition, people with this two risk single genotype have the highest hypertension (1.5), high triglyceride (1.9), high blood pressure combined with high acid triglyceride (2.3) and metabolic syndrome ( 1.8) Hazard comparison value. First, our results indicate that the two common single sets of genes are associated with hypertension, sputum sputum, high blood pressure combined with TG, and metabolic syndrome. We used the initial linkage analysis of satellite markers and the identification of related studies using a single set of genotypes using L-SNP markers, with strong support for direct participation. In patients with two B (C/AA/T) and D (GTAG) risk single genotypes, 'four metabolic syndromes are the most at risk, and the proportion of these two single genotypes is not low. . 1357442 All, the risk of a single set of base _ is a common variation, the division = early sets of base 2 dirty m 1 by Claric et al. Gasol and African American data from the '88 sets of genes Lieutenant % BmΒ risk single set of genes. Regarding Bin D, in the same research = 12 sets of bases rely on 88 single sets of bases. Therefore ^ = early genotypes are common in the country of the country, but also f-class high-plus New Zealand = ^ to about the news _ offer. The average relative risk of the syndrome is about 15%_p(10)33. The average relative risk of the syndrome is about 15% _p(10)33. It shows that the stage = 15% of the metabolic syndrome phase _ can be attributed to two J = PL early genotypes, therefore, we have identified a common occurrence for common complexities, hypertensive metabolic syndrome Genetic variation. Xie Guangitr's LPL mutation from Newton has been described in a variety of lipid-like metabolic diseases, but most of these are rare mutations, including 61 missense mutations, 12 nonsense mutations, and 1 frame transfer. Mutation or mini-insertion/deletion, 3 non-spot mutations, 8 splicing mutations (splidng __ and 4 promoter variants (Merkel M et al.), in which 35 The mutations are located in the fresh 4, 5, and 6 series outside the LPL, that is, the areas where B, C, and D are recognized. According to Tilbeurgh H van, (1", 36, LpL protein has two distinct regions: N The end range (amino acids, encoded by exons 1-6) 'is responsible for enzyme catalysis, and the c-terminal range (amino acids 313_448, encoded by exons 7-9), which binds to the lipoprotein matrix 丨^^^, 2〇〇2)12. These two ranges correspond to two Z/2: unbalanced junction blocks, which are located in the intron 6, the i.9kb of the parent of each child 6 in the near-outer member 6. The recombination hotspots are separated (Templeton AR et al, 2000) 37. Therefore, the melon B and the candied melon variation are located in intron 3, respectively. · 4 and intron 5_6, its ploughing is related to the performance function of LpL. 1357442 No mutation was found in the promoter region (the proximal 571 bp tested) and the LPL gene except for a SNP located in exon 8. And a Ser44X7 located in exon 9 Although the protective single genotype of exon 8-9 has a lower than expected delivery profile, which is sufficient to affect the high diglyceride and progeny of hypertensive patients, the degree of correlation Not as strong as intron 5-6 (determined by transmission imbalance test), and it is not related to = blood pressure risk and other metabolic syndrome-related phenotypes (data not shown). ° Generally considered to be located within one The polymorphism in the region of the exon may be linked to an imbalance of functional mutations at the junction of the coding region or the intron/exon. However, we identified this two-risk single genotype. It is not within the imbalance of mutations outside of any cause. To understand how these intrinsic variations σΑΑ/τ(ς, exon 4) and GTAG (cross exon 6) work, further testing is needed. At least six splices (splic_ma l) The different functions of introns are proposed 'including (1) non-coded RNA sources; (2) vectors for transcriptional regulatory elements; (3) selective and trans-splicing methods (trans_splidngW performers; (4) coding sequences minus The promoter of the meiotic crossing; (5) the matrix of the exon-mixing; and (6) the output signal of the nucleus and the regulation of the decay (Fed L 咖 咖 咖 咖 咖 咖 2003 〇 2003 2003). The correlation between the genotype of melon and the local blood pressure and metabolic syndrome can be known, solution, metabolic syndrome and obesity and the resistance of Tengdaosu _, hyperglycemia, hyperemia, oily day, and less Dong Density lipoprotein cholesterol is a metabolic syndrome. The various aspects of the metabolism of these axes are related to each other, and the pathogenesis or order of occurrence of dysfunction, lipotoxicity and obesity may be more than two. Triglycerides are involved in the accumulation of fluids and tissues, which may lead to hypertension, high triglycerides = metabolism. The literature documents many other functions of LPL, such as increasing, bridging the function of glutinous proteins, a monocyte-binding protein, and promoting the proliferation of smooth muscle cells, including tumor fiber alpha factor Gene expression, stimulation of acid oxide synthase expression, activation of nicotine guanamine adenine dinucleotide / alkali guanamine adenine dinucleotide phosphate (ΝΑΕ^ΘΗ) oxidase and reduction of apoprotein The secretion of Αρ〇Ε) (Mead et al, 2002) 12, many of these mechanisms can also contribute to the formation of hypertension. In general, the present invention provides a method for predicting a patient's genetic risk of hypertension, high triglycerides or metabolic syndrome, including screening for a patient's gene. The emergence of a single genotype of C/AA/T or a single genotype of GTAG or both is a risk indicator for metabolic syndrome-related conditions. The gene sequence can be investigated by any method established in the art, for example, using an oligonucleotide that specifically binds to a single set of gene nucleic acid sequences. Polymerase chain reaction (PCR), polymerase chain reaction combined with single-strand configuration polymorphism analysis (PCR-SSCP), denaturing gradient gel electrophoresis (DGGE) and nuclear enzyme A dislocation truncation method are several methods of the existing methods An example. It is worth noting that in addition to the single set of genes and SNPs mentioned here, genetic markers linked to any of the different sets of genes and SNPs can also be used to predict risk. This is because the genetic markers of a single set of genes and SNPs close to these targets will be separated from the target set, and will be separated or unbalanced due to SNPs. Therefore, the appearance of these markers (with genetic markers such as φ) indicates the presence of a single set of genes and SNPs, which indicates the risk of high blood pressure, high diglycerides, and/or metabolic syndrome. Equivalent to the mark. The mark can be any mark, including microsatellites and SNPs. Preferably, the effective genetic marker is derived from the ZPZ gene, which is about 200 kb or less. More preferably, it is derived from the LPL gene and is about i〇〇kb, 80kb, 60kb, 40kb, 20kb in size:

L^kb or 5kb or less. Unbalanced coupling can be determined by well-known methods in the art, including the methods described herein. /N The invention also provides nucleic acid fragments (single or double strands) comprising the SNPs described herein or a single set of genotypes. This fragment can be used as a probe. Primers that can be used to detect single (tetra) acid genotypes are also provided. Nucleic acid fragments or primers can be used in various groups 12 1357442. For example, detecting a set of genes

Probes or primers for three SNPs of C/ΑΑ/Τ. (4) Early morning lane U is the same as the probe or primer for the four sets of genotype GTAG four SNPS, ϊίίί--crowd. The lion (4) can include other products, such as: • Heterogeneous hybrid equipment and/or reagents, polymerase chain reaction (PCR) equipment and/or reagent primer extension reagents, equipment and/or reagents for collecting peripheral blood. Nucleotide primers or probes are used as instructions; and • Reagent containers are provided as a method for pharmacogenomic analysis. Therefore, 疋SI human-partial genetic factors, and each genetic factor*, is associated with a predisposition. According to the present study, a single set of genotype C, /AA/, T, and genotype GTAG was targeted for hypertension, and early markers. For the same sputum and heart - glycerol sputum southerly or metabolic syndrome in addition to Na 4 胄 - Λ ’ or 柯 selectively contain other genetic factors. It may also be included in the sputum or in contact with any other known side. The following examples are provided to further illustrate the present invention and the following examples of the embodiments of the invention. Unspoken abbreviations have their usual meanings. 1357442

°c ~ Celsius temperature hr = hour min = minute sec or s = second μΜ = micromoles mM = milliamps M = mol ml ml = ml μΐ = microliters mg = = milligrams pg = micrograms mol = moles (mole) pmol = picomol LPL HTN TG MS SBP DBP SNP = = mononuclear acid polymorphic PBS : = phosphate buffer solution NTP = three-packed nucleic acid PCR = polymerase chain reaction NPL = non-parametric junction ASO = Specialized oligonucleic acid

14 1357442 Materials and methods (1) Patient and family (A) Initial linkage and related studies 21 (H-solid hypertension clinical patients collected from 2 North Rongmin General Hospital, from a family of early-onset hypertension cores. In the towel, there are 25 brothers and sisters (pan, poor 2000). For the initial linkage analysis, the sample has 7 microsatellite marker genotypes near the hunger. In addition, 9 triglycerides were collected later. A high-risk (>= 150 mg/dL) early-onset high-inflation family, a total of 267 subjects were used for the identification of the 'and the construction of a single genotype, and used for the family-related stone of the next step if (B) Family-related confirmation study The study collected data in three Hakka areas in northwestern Taiwan. The Hakkas were Chinese Han Chinese immigrants from Taiwan to Guangdong Province. The prognosis of early-onset hypertension ^ ^obandj comes from In 4 community hospitals, for each confirmed proband, his/her members of the court were subjected to blood pressure screening and health screening. A total of 345 ethnic groups and 1002 family members were included. , 384 early-onset hypertension parent-child φ trio ( Trios) was used to confirm the study. The Human Investigation Committee of the Institute of Biomedical Sciences of the Academia Sinica has approved the procedure for this study. (2) The definition of hypertension and related phenotypes are defined as any of the following conditions, defined as high Blood pressure: (1) systolic blood pressure (SBP) at rest is higher than or equal to 140 mm mercury column, (2) diastolic blood pressure (DBP) is higher than or equal to 9 mm mercury column, and (3) antihypertensive drugs are currently taking. Hypertensive patients under the age of 40 are defined as early-onset cases. In addition, we also use two other sub-phenotypes in family-related studies, (!) hypertension combined with triglyceride High and (2) metabolic syndrome. We define high triglyceride with a fasting triglyceride value higher than or specific to 150 mg/dL, and define metabolic syndrome as defined by the modified NCEp_ATp m 2002)21. If at least the following three conditions are met, it means that a person has metabolic syndrome: triglyceride ^15〇mg/dL, man's high-density lipoprotein cholesterol (HDL-C) <40 mg/dL/ woman's face street Lipoprotein cholesterol < 5 〇 mg / dL, higher body mass index (227), hypertension (systolic pressure ^ 135, diastolic pressure ^ 85 mm mercury column or taking antihypertensive drugs), and fasting blood glucose > 110 mg/dL (or receiving diabetes treatment). This study used the Taiwanese obese body mass index standard (see Pan et al., CAA 2〇〇4). (3) Initial linkage and correlation analysis with microsatellite marker genotypes The peripheral lymphocytes of 59 probands and their 151 relatives were collected by phenol/gas imitation extraction method and isolated to obtain their genomic DNA (Aggarwaletal. , 1992) 23. There are eight microsatellite marker polymorphisms at chromosome 8p22, including D8S1731 (31.73cM), D8S261 (37.04cM), D8S1145 (37.04cM), GZ-14/GZ-15 (GDB:177387 or LPL markers; 39.25 cM), D8S322 (41.55cM), D8S1116 (42.85cM) and D8S382 (51.15cM). The genotyping steps for microsatellite markers can be found in the previous report (pan et al, 2000). Fragment analysis was performed on an ABI377 DNA sequencer and analyzed using GeneScan version 3.0 and genotyper version 3.0. Dual gene detection (aueie caning) and reconfirmation were performed independently by two individuals. (4) Discovery and confirmation of single nucleotide polymorphisms (SNPs) of LPL genes The LPL gene has 10 exons and spans approximately 30 kb on the short wall of chromosome 8 for the purpose of accessing the database (http: //www.ncbi.nlm.nih.gov/snpdatabase) To verify the identified SNPs and discover more new SNPs in the Chinese Han nationality, we will use the promoter region (to contribute to the positive npl score in the early onset hypertension family) The most 1357442 - 41 people), the extensor 1-9 (41 subjects) outside the muscle-striped horse area, and the selected 571 bases of the captured introns of the published SNPs (4) Job ^ over 1998) 24. Then we will have a 13 lion, located in the LpL region, containing only a few known SNPs, introns 2, and intron 3, which are sequenced from 12 subjects (6 early onset) Sexually high blood-blooded patients and 6 normal blood pressures with age and gender. (5) Unbalanced Linkage Mapping of SNPs Genotypes and Related Studies In order to establish an unbalanced linkage (LD) map within the gene, newly identified SNPs and confirmed SNPs were obtained from DNA from 267 Chinese Han Chinese. Mas? ARRAYSNP genotyping system (8 Ε (3 υΕΝ〇Μ) analysis determined (Table 2). Polymerase chain reaction using 20 μl of l〇ng gene DNA, one

Perkim Elmer GeneAmp 9700 (Foster City, CA) thermal cycler. The final reagent concentration is 1x AmpliTaqGold PCR Buffer (PE/ Applied)

Biosystems, CA, USA) contains 2.5 mM MgCl2, 0.2 mM dNTP, 〇.2μΜ various primers and 0.4UAmpliTaqGold. The initial denaturation was carried out at 94 ° C for 15 min, followed by 45 denaturing cycles of 20 s at 94 ° C, 30 φ s bonding at 56 ° C and 1 min extension at 72 ° C. . The final extension was carried out at 72 ° C for 3 min. The extension of the PCR product is extended by the SNPs.

Homogeneous MassEXTEND spectroPREPTM (SEQUENOM) is executed. The nine SNPs representing Bins B, D and F as shown in the first figure were selected from the SNPs genotypes of 1002 subjects that were confirmed to be studied. The second SNP at BinB is polymorphism resulting from insertion/deletion. (6) Statistical analysis For the initial study, data on 25 pairs of fraternal pairs of rickets in 59 families were analyzed by multi-point linkage analysis using the GENEHUNTER program (Kmglyak et al, 17 5 1357442-1996) 25. In addition, information on 42 patients with early-onset hypertension and their mothers from 35 families was used to perform the paternity transmission imbalance test (Spidman and Ewens' I"8)26. The Ponferroni test (B〇nferr〇ni • Procedure) is used to adjust multiple comparisons26. The generalized estimating equation (GEE Version 2, 〇 3) (Liang KY et al, 2001) 27 uses clinical chemistry data, including fasting triglycerides, to compare the phenotypes between early-onset hypertension and unaffected close relatives. In order to establish an unbalanced binding (LD) map of the LPZ gene, 44 confirmed SNPs with a minor gene frequency (MAF) of 20.05, and their linkage disequilibrium blocks were estimated by GD (Graphical Overview of Linkage Disequilibrium) (Abecasis et Al, 2000) 28. Consider the following parameters to select and classify SNPs: information on linkage disequilibrium blocks (D'>0.8), whether MAF is greater than or equal to 〇j, and whether they are importantly associated with combined SNPs and explicit SNPs. Use TRANSMT, Logic (2.5.2, April 2000) (Clayton and Jones, 1999) 29 to perform a transmission imbalance test for a specific set of genes. One analysis of bootstraps was performed in this analysis. To estimate the predicted value of a single set of genotypes of SNPs for predicted hypertension and related phenotypes, use the SIMWALK program (version 2.86, July 2003) ( Sobel,

Lange, Aw. Qiu Ge Ge« said 1996) 30 identified the SNP single genotype of each individual in the study. A variety of 8 see > risk comparison values for a single set of genes were estimated using the (} £ £ method, SAS (SAS release 8.2) software. Example 1 Linkage and correlation analysis between microsatellite markers and early-onset hypertension Multi-point linkage Analysis showed that there is a linkage position of early-onset hypertension in the vicinity of gene intron 6 (GZ-14/GZ-15), with a npl index of 3 (independent independent relatives with a logarithm of 25) To the signal. In addition, by

Spielman's parent-child parental imbalance test (s_D8S322 has significant correlation (Table 1). The capture of the marker and the D8S322's Chegan () hunger-to-do dual gene 1 (p=1.5 X 1〇7) :==== .8 x — handed to early-onset high progeny than off!. As with other dual genes. Therefore, this analysis shows that (10) is associated with hypertension, one marker (cM), NE-based, and the same. Index - _ X5 Ϋ§~ Number of ρ values D8S1145 37Ό4 56 28

LPL 39.25 55 15 17 3 2.04 1.33 7.39 0.04 0.18 1.5χ1〇·13: 0.34 3.02 D8S322 41.55 49 J 1 38 % 4 24 2+ 38 4 6.23 3.05 5,50 4.6χΐ〇·1〇ϊ 0.002 3.8Χ10'8* 1.38 D8S1116 42.85 47 4 11 1 4 37 9 D8S382 51.15 45 3.49 1.68 1.75 0.0005 0.09 0.08 1.19 0.27 heart ^ couple machine f does not give birth to this phenomenon) * Ρ <1〇-5 the number of this AM generation (when other pairs of machines Because of this phenomenon) %>L·Adjustment of multiple markers and dual genes for nonparametric pairs of ϋ-linked analysis 19 1357442 Example 2 Comparison of clinical symptoms between high-pressure patients and normotensive patients: an initial correlation study The clinical findings are consistent with the above results. The fasting diglyceride concentration in male patients (41 mg/dL, p=0.017 and female 33 mg/dL, p=〇.〇4) is significantly higher than the same age, gender, and Normal control group of residential area and body mass index (OienJW, /«/Ja^W, 2004). In the confirmed study, the concentration of fasting triglyceride and most of the metabolic syndrome related to hypertensive patients and unaffected close relatives contained body mass index, high-density lipoprotein cholesterol, cholesterol, fasting tamsin and blood sugar. The difference (Table 2). Table 2 Comparison of traits related to metabolic syndrome in early-onset hypertension cases and unaffected relatives

Hypertensive cases unaffected in close relatives t P value * ___ average (953⁄4 CI) Male (224) female (155) male (161) female (176) Age (years) 39.7 ± 6.2 40.8 ± 5.5 42_1 ±9.0 41.8±9.4 Systolic pressure (mm mercury column) 139±18 137±18 124±17 118±18 19 (16.6-21.9) <0.0001 Diastolic pressure (mm mercury·column) 92±13 88±12 79±12 75 ±12 14 (11.9-15.6) <0.0001 Body Mass Index (kg/m2) 27.4±3.9 26.0±4.4 25.1±3.1 23·9±3·8 2.2 (1.7~2,7) <0.0001 Waist circumference (cm) 91.9 ±10.0 82.7 ±10.3 86.4±8.9 76.6 ±9·8 6.3 (4.9-7.7) <0.0001 Triglyceride (mg/d〇223±216 156±174 176±156 121 117 47 (25.1-69.0) &lt ;0.0001 high-density lipoprotein cholesterol (mg/dl) 43 ±10 52±12 47±12 55 soil 14 •2 (·4·2~-0.7) 0.007 low-density lipoprotein cholesterol (mg/dl) 129±51 125 Soil 33 124±32 116±33 9 (2.9-15.0) 0.004 1357442 Cholesterol 〇ng/dl) 211±58 202±42 200±40 191±36 14 (7.05-21.1) <0.0001 Insulin (IU/ml) 18士24 16土16 14±14 12±8 3 (0.9-4.8) 0.005 glucose (mg/dl) 115士48 11 6±53 102±32 105±59 11 (3.04-18.6) 0.007 ^Study=Use 345 household data for mixed models to control family relationships, and make gender and age as co-variation Affected reference group values are used to compare logarithmic conversion values

Genes and constructs a single set of genes within a single nuclear polymorphism of the unbalanced linkage map in order to identify a single set of genotypes associated with hypertension risk and other metabolic syndrome-related symptoms '44 of the MAF in the gene is greater than 0.05 The SNPs (Table 3 identified two major unbalanced maps for the DNA of 267 individuals. We constructed six bins in the 30 kb region of our gene: four ld PCT at 5, Bins AD ), and two LD blocks at the 3' end (Bins E_F) (first TD. Among them, the variation on BinsB, D and F is related to hypertension.'

Figure-b shows the most common single set of genes in Bins B, D and F. Gold vs. Bm B (intron 3-4)' shows three polymorphisms (s Pengzhou, SNP 2 and SNP6 53). The single genotype of the most common m in Bin B is c/aa/t, and the target is 1Bin D (intron 5·6) 'risk phase_single set of genes p S 〇"13,^^,J^AF050163 -25〇〇'rs269^rs27( and rs 1). Risk-related SNPs 30 and 38 (rs328) for Bm F (exon 8_9) towels. Dry to 丞ui two...(方, 5 21 1357442 Table 3: Polymorphism of lipoprotein lipolytic enzyme gene in Taiwanese population and polymorphic polymorphism of its dual gene frequency _ (dual gene frequency yi . Gene bank NT 030737 tag name tag _ 爽 location number ~ ending at NT_030737* in Taiwanese

LPL-1322 7606407 7606407 — A/G (0.96/0.04) A(10) A P1-1 7606596 7606596 G/T (rsl470187) G(1.0) X P1-2 7606710 7606710 T/C(rs 1470186) T(1.0) X LPL -53 7607528 7607528 — C/A (0.83/0.17) C(1.0) Exon 1 7607873 7607960 — 7608249 7608249 G/T (rs2898492) G(T〇) x ... 11-2 7608837 7608837 G/T (rs3779787) G/T (0.71/0.29)* X 11-4 7610208 7610208 — A/C (0.96/0.04) X LPL-55 1 7610562 7610562 (rsl534649) G/T (0.71/0.29)* G/T (0.89/0.11 11-7-1 7612033 7612033 G/A (rs 1031045) G(1.0) X LPL-1323 7612451 7612451 — T/G (0.67/0.33) X 11-9 76i3323 7613323 — C/T (0.71/0.29)* X 11-10 7613481 7613481 — G/A (0.96/0.04) X 11-11-1 7613779 7613779 — A/C (0.96/0.04) A (1.0) 11-11-2 7614014 7614014 C/T (rs3779788) C /T (0.71/0.29)* X 11-13-1 7614752 7614752 — G/A (0.96/0.04) X 11-13-2 7614917 7614917 One T/A (0.71/0.29)* X 1 Exon 2 7616612 7616772 — LPL-E2 7616629 7616629 Asp9 an Asn G(H) factory...—... LPL-186811 2 7617592 7617592 C/G (rs8176337 C/G (0.71/0.29) C/G (0.89/0.11) Exon 3 7620201 7620380 — LPL-E3-1 7620215 79620215 Tyr61—stop T(lO)" — LPL-E3-2 7620356 7620356 G/ A (rsl 121923) G(1.0) 7621122 7621122 T/C(rs8176338) T/C (0.96/0.04) T/C (0.99/0.01) LPL-1871 3 7621708 7621708 C/A(rs343) C/A (0.79 /0.21) C/A (0.87/0·13) 7621712 7621712 A/C (rs247) A (1.0) X ; exon 4 7621742 7621853 — •~. LPL-E4 7621747 7621747 G/A(rs248) G( 1.0) rs249 4 7621927 7621927 T/C (rs249) T/C (0.94/0.06) T/C (0.93/0.07) P4 1 1 7621945 7621945 T-/TG/GT (rs250) T(IO) T(1.0) 7622081 7622081 T/C(rs251) T(1.0) X P4 1 23 5 7622153 7622153 A/· (rs252) AAA/AA AAA/AA (0.75/0.25) (0.63/0.37) P4 1 4 6 7622338 7622338 T/C (rs253) T/C (0.6/0.4) T/C (0.61/0.39) Outer buckle 5 7622552 7622785 — LPL-E5-1 7622617 7622617 G/A (Ala176—Thr) G(1.0) LPL-E5-2 7622654 7622654 G/A (Gly188^Glu) G(1.0) LPL-E5-3 7622672 7622672 T/C (Ile194^Thr) T(1.0) LPbE5-4 7622703 7622703 C/G (Asp2°4—Glu C(1.0) P4 2 1 7 7622818 7622818 C/G (rs254) C/GC/G (0.83/0.17) (0.625/0.375) tf P4_2_2 7622822 7622822 T/C (rs255) T/C (0.625/0.3 75 ) n X 7622888 7622888 C/T (rs256) C(1.0) X 22 1357442

Marker name tag number t at the NCBI gene bank NT_030737 position polymorphism polymorphism begins at NT-030737* (publication > ^) in Taiwanese t (dual gene frequency) (dual gene frequency) n 7623149 7623149 A/C (rs257) A (1.0) X P5_l 8 7623173 7623173 G/C (rs258) G/C (0.5/0.5) G/C (0.60/0.40) 7623358 7623358 A/T(rs259) A (1.0) X 7623420 7623420 C/G (rs260) C(l.〇) X 7623541 7623541 A/GA (1.0) X (AF050163-1939) 7623563 7623563 ATT A (1.0) X (AF050163-1961) P5 a 2 9 7623733 7623733 C/ TC/T (0.65/0.35) C/T (0.73/0.27) (AF050163-2131) P5 - 3 10 7624101 7624101 G/AG(1.0) G/A (0.82/0.18) (AF050163-2500) rs265 7624190 7624190 C /G (rs265) XC(1.0) rs266 7624220 7624220 A/G (rs266) XA (1.0) rs267 7624239 7624239 C/T(rs267) XX Exon 6 7624273 7624515 — ; LPL-E6-1 7624359 7624359 G/A (Ala261—f lir) G(lO) LPL-E6-2 7624450 7624450 A/G (Asn291—Ser) A (1.0) LPL-1325 11 7624588 7624588 T/G (rs269) T/G (0.64/0. 36) T/G (0.83/0.17) 7624589 7624589 G/T (rs2075651) G(1.0) X LPL-56 12 7624597 7624597 C/A (rs270) C/A (0.91/0.09) C/A (0.82/0.18 LPL-57 13 7624623 7624623 G/A(rs271) G/A (0.64/0.36) G/A (0.82/0.18) 7624849 7624849 C/G (rs272) C(1.0) X 7625154 7625154 C/T (rs275) C(1.0) X 7625210 7625210 T/C (rs276) T(1.0) X LPL-58 14 7625444 7625444 — G/A (0.91/0.09) G/A (0.82/0.18) 7625617 7625617 C/G (rs279) XX 7625803 7625803 G/A(rs280) G(1.0) X LPL-59 15 7625944 7625944 A/T(rs281) A/T (0.77/0.23) A/T (0.72/0.28) LPL-60 7625947 7625947 C/G ( Rs282) C(1.0) C(1.0) LPL-61 7625968 7625968 — T/C (0.96/0.05) T/C (0.99/0.01) LPL-62 16 7626019 7626019 CAT (rs283) C/T (0.82/0.18) C/T (0.80/0.20) LPL-63 7626027 7626027 T/C (rs284) T(1.0) T(1.0) LPL-64 17 7626110 7626110 C/T (rs285) X CAT (0.63/0.37) LPL-65 7626177 7626177 A/T (rs286) XA(lO) LPL-130 18 7626477 7626477 A/G (rs6586882) A/G (0.75/0.25) A/G (0.83/0.17) 7626522 7626522 G/A(rs2583659) G(1.0 ) X LPL-131 19 7626540 7626540 T/C (rs2853630) T(1.0) T/C (0.84/0.16) LPL-132 7626759 7626759 G/A(rs2698206) G(1.0) G(1.0) LPL-133 20 7626773 7626773 T/C(rs291 T/C (0.7/0.3) T/C (0.92/0.08) 7626976 7626976 G/A(rs292) G(1.0) X P8 2 1 7627000 7627000 A/- (rs293) A/-(0.0/1.0) X P8 2 2 7627046 7627046 T/C (rs294) T(1.0) X P8 2 3 21 7627159 7627159 A/C (rs295) A/C (0.94/0.06) A/C (0.85/0.15) 7627165 7627165 G/A ( Rs296) G(1.0) X P8_2_4 7627292 7627292 T/C (rs297) T(1.0) X Outer 7 7627692 7627812 — LPL-E7-1 7627781 7627781 G/A (rs298) G(1.0) LPL-E7-2 7627801 7627801 C/T(rs299) C(1.0) LPL-E7-3 7627808 7627808 A/G (rs300) A (1.0) LPL-66 22 7627855 7627855 T/C(rs301) T/C (0.68/0.32) T/C (0.83/0.17) 23 1357442 in the NCB1 gene bank ΝΤ _030737 polymorphism

The position polytype of the tag name tag number t begins to end at ΝΤ_030737; (public i"called) in Taiwanese (dual gene frequency) (dual gene frequency) 11 LPL-67 7627888 7627888 C/T (rs302) C( L.〇) X LPL-134 23 7628200 7628200 G/C (rs303) G/C (0.95/0.05) G/C (0.91/0.09) LPL-135 24 7628282 7628282 T/G (rs 1963909) T/G ( 0.7/0.3) T/G (0.83/0.17) LPL-136 25 7628322 7628322 A/G (rs2244603) A/G (0.7/0.3) A/G (0.83/0.17) LPL-137 26 7628397 7628397 — T/G (0.85/0.15) T/G (0.93/0.07) LPL-138 27 7628467 7628467 C/T (rs2853631) c/τ (0.90/0.10) C/T (0.91/0.09) LPL-150 7628543 7628543 AAAT/- ( Rs311) AAAT (1.0) AAAT (1.0) LPL-68 28 7628918 7628918 G/C(rs312) G/C (0.92/0.08) G/C (0.91/0.09) LPL-69 7628947 7628947 A/G(rs313) A (1.0) X LPL-70 29 7628963 7628963 G/A(rs314) G/A (0.63/0.38) G/A (0.83/0.17) LPL-71 7629016 7629016 T/C(rs315) T(1.0) X Sub 8 8629333 7629515 LPL-E8-1 30 7629357 7629357 C/A (Thr361—Thr) C/A (0.83/0.17) C/A (0.91/0.09) LPL-E8-2 7629 472 7629472 G/A(Ala400—Thr) G(1.0) Rs317 31 7629692 7629692 AG/-orGn"(rs317)x G/T (0.91/0.09) LPL-72 7629890 7629890 C/G(rs318) C(1.0) C(1.0) LPL-73 32 7629897 7629897 A/C(rs3l9) A/C (0.86/0.14) A/C (0.80/0.20) LPL-74 33 7629998 7629998 T/G (rs320) T/G (0.68/ 0.32) T/G (0.83/0.17) LPL-75 7630107 7630107 G/C(rs321) G (1.0) G(1.0) LPL-76 34 7630138 7630138 A/C (rs322) A/C (0.68/0.32) A /C (0.83/0.17) 7630142 7630142 A/C (rs323) A (1.0) X 7630143 7630143 A/- (rs324) A (1.0) X LPL-77 35 7630249 7630249 T/C (rs325) T/C (0.73 /0.27) T/C (0.92/0.08) LPL-78 36 7630360 7630360 A/G (rs326) A/G (0.68/0.32) A/G (0.83/0.17) LPL-79 _37_ 7630457 7630457 T/G (rs327 T/G (0.70/0.30) T/G (0,83/0.17): outer tweezers 9 7630547 7630651 * * * ..............- LPL-E9 38 7630645 7630645 C/G (Ser447—stop) C/G (0.95/0.05) C/G (0.92/0.08) (rs328) 7631007 7631007 A/G (rs329) A (1.0) x LPL-80 39 7631317 7631317 G/A ( Rs330) G/A (0.92/0.08) G/A (0.91/0.09) LPL-81 40 7631326 7631326 G/A (rs331) G/A (0.92/0.08) G/A (0.83/0.17): Well-being 7633742 7635690 1 (3' untranslated area) Ρ13-1 7634113 7634113 T/C (rs3289) T/C ( 0.96/0.04) T/C (0.996/0.004) 7634567 7634567 G/A (rs 1059497) G(1.0) X LPL-1324 41 7634569 7634569 A/T (rs3208305) A/T (0.56/0.44) A/T ( 0.84/0.16) Ρ13 33_1 42 7634595 7634595 _ C/T (0.73/0.27) C/T (0.9/0.10) Ρ13_3_2 7634884 7634884 C/T (rsl059507) C/T (0.95/0.05) x P14_l 43 7635413 7635413 T /C (rsl 3702) T/C (0.73/0.27) T/C (0.86/0.14) Ρ14_2 44 7635484 7635484 T/C (rs 1059611) T/C (0.77/0.23) T/C (0.92/0.08) Ρ14_3 7635588 7635588 C/T (rsl 5285) C/T (0.71/0.29) X Ρ14_4 7635600 7635600 C/A (rs386647) C/A (0.96/0.04) X Ρ14 a 5 7635789 7635789 C/A(rs3916027) G/A (0.71/0.29) X: The sequence number of a single nucleotide polymorphism for LD and TDT analysis is shown in Figure 1 and Table 3 -

J. The disclosed single nucleotide polymorphism for variation was obtained from NT_008081 (2001 April version) and Ντ 〇〇827丨 (2〇〇1 October version) *> 1. 12 Taiwanese used for sequencing Samples (6 hypertensive patients and 6 normal blood pressure control groups) _ However, for the external; 罝_4钵赭 polymorphic 'samples from 41 targets of the early-onset hypertension family (see method). " Use ^ to determine the frequency of the dual gene, which is the 267 targets in the initial study. *, the single nucleotide polymorphism with this symbol is completely unbalanced with the other. 24 442 i^8^^^337) and LPL]87Q (4) 176338): These single-(four) shirts are sent to the NCBI's single nuclear hair--: non-polymorphic; X: bit is set genotype embodiment 4 transmission is not Correlation between single nucleotide polymorphism in equilibrium analysis and phenotypes associated with early-onset hypertension and other metabolic syndromes. In the initial study, 267 cases were analyzed and found in a single set of genes and high in the validation study. Correlation of blood pressure imbalance transmission analysis, the results obtained were consistent. In the confirmation study, 15 to 5 case data were used (expanded by the poor materials of the original 1〇〇2 individuals) (Table 4). High for triglycerides (p=〇〇〇5), high for triglycerides for hypertension (Ρ<0·0001), and for metabolic syndrome &=〇〇3), Bin B The proportion of genotype C/AA/T delivered to progeny was significantly higher than the others. Conversely, for the combined phenotype of sputum blood pressure and diglyceride, the single set of genotype C/AAA/C of block B was passed to the offspring _ lower. Regarding Bin D, for the high phenotype of hypertension combined with triglyceride, the single genotype GTCG & GTAG was transferred to the offspring, which were significantly lower and higher &<〇〇〇〇1) A single set of gene-side GTAG in Bm D 也 was also found to be positively correlated with hypertension and metabolic syndrome, but to a lesser extent. For BinF, a single set of gene CG is targeted for high levels of triglycerides and high blood pressure, which is transmitted to fewer offspring (〇4 P=0.03). ‘ 1357442

$ ^ HQH ^ 1^ ^s,, ^.ί ^ , 4^$sws0 βοοο^ s< \~¥ 〇. 0.269 0.034 0.067 0.037 0.446 0.208 0.076 0.094 Β in F (single nucleotide polymorphism 30,38 t ν〇Ο $ 2 407 58 § ^ Obs S - Iso-ON 415 50 g - Single set of genes α α υ υ 8 8 uauu U 〇UUZ 253 2 (Ν BinD single nucleotide polymorphism 10, 丨 1, 12, 13" 〇. 0.149 0.049 0.330 0.062 <0.0001 * <0.0001 * 0.720 0.022 ^ CN ν〇IT) — 319 75 R - Obs CN «•/Ί 对^Τϊ — 312 92 Single set of genes GTCG GTAG GTCG GTAG GTCG GTAG GTCG GTAG 2 435 2 οο Γ^ί (Ν Bin B (single nucleotide polymorphism 3,5,6)1 Q. 0.665 0.176 0.485 0.005* 0.007* <0.0001* 0.875 0.033 卜 00 m οο m — 〇〇in Os O (N — a 卜σ\ (Ν 00 ο CN — Obs iT) ON — 294 118 cn (NO (N — οο γλ οο — (Ν — Single set of genes § C/AAA/CC/AA /TC/AAA/CC/AAJT C/AAA/CC/AA/TC/AAA/CC/AA/TZ 427 243 § (Ν m (Ν 卞 卞 卞 血 血 三 三 三 ώ 压 压 压 压 压 压Triglyceride is a metabolic syndrome. ϊ — - * Forget Dare sE: dx3ii Dang Ja > wheel braking benches not call back Yasushi continued # # Xu v ^ sqom ^ s benches drums manufactured by: z ____- ¾0 ^ - ^

5 1357442 Example 5 Single set of genetic risk comparison values for hypertension and other metabolic syndrome-related phenotypes The relative risk or clinical risk for a single set of genotypes C/ΑΑ/Τ for the estimated Bin B is 1.5 (p= 0.007) (hypertension), 1.5 (p=〇.〇〇8) (different triglyceride), 1.8 (p=〇.〇〇〇l) (hypertension combined with triglyceride) and 丨3 φ =〇〇8) (metabolic syndrome). For the above clinical results, the risk comparison values of Bin D's single genotype GTAG were 1.5 (ρ=〇·〇2), 1.7 (p=〇.〇〇i), and 1 9 (ρ<〇.〇〇〇) 1) and 1.2 (p 0.002) 〇 BinF's single genotype hazard comparison value is not associated with an increased risk of any of the above clinical outcomes. In addition, I took a single set of genetic status of Bins B and D, and Γ ί ΐ four seed groups. It has a high risk ratio of any single gene set in Bin B or Bin D risk. Material group) (second picture). · One, Ό D ) and 1.8 (for the case of a metabolic bed, the results are changed to 6 and 5 with higher or lower, homologous individuals with introns 4, 5 and 6 homologous individuals) The genetic structure of the lipolytic enzyme gene. & BlnsB to D) 'Single set for confirmation and inference 27 1357442 [Simplified illustration of the drawings] The first figure illustrates the relative positions of genes and SNPs in the components studied in the present invention. The first panel (a) shows the API gene map. Nine exons, E1-E9, are represented by vertical solid bars labeled E1-E9. Bin A-F is shown in a solid box with the letters A-F below. The SNPs 1-40 position (see Table 3 for a detailed description of SNPs) is shown in parallel lines above the LPL gene pattern. Figure 1 (b) shows the sequence of a single set of genes commonly found in Bins B, D and F. This percentage indicates the percentage of occurrence of each individual set of genes in the study population. The second panel shows the hazard comparison values for the single genotypes of Bin B and Bin D for hypertension, high triglyceride, high blood pressure, high triglyceride (HTN-TG) and metabolic syndrome. For example, the rightmost column describes the hazard comparison of Bin B's C/AA/T single genotype for metabolic syndrome. When Bin D is GTAG, the hazard comparison for individual genotypes is 1.8. When Bin D is GTCG, the hazard value is 0.9.

28 5

Claims (1)

1357442 Patent application & annual body repair (more) original ^Β@Ί〇Ο^Ι2月19 Report 'This 1. A method for predicting the risk of hypertension and triglyceride deficiency syndrome in patients' This includes investigating the patient's lipoprotein lipolytic enzyme (the Seto Mirage Sequence) and the aforementioned gene sequence is selected from the following IPX single sets indicating the risk of hypertension, high triglyceride or metabolic syndrome. Gene group: , , ', (a) a single set of genes C / AA / T, which contains c located in rs343, AA in rs252, and T in rs253; and, (b) a single set of gene GTAG, It contains G located at AF050163-2500, T at rs269, A at rs270, and g at rs271. 2. ^Special fiber (4) 1 Lie's green, its cut set gene is 3. ίτ^ full-time (four) 1 nar The branch, the gene of the cutting set is the first one, and the method of cutting the gene contains one item of the method ^ wherein the aforementioned sequence is stored by using the 'degree of strictness' and the specific impurity 11=profit_the first item In the method described, the delineated sequence is investigated by using DNA prepared from human peripheral blood. ', 曰The method according to the above item 1, wherein the sequence is detected by using sputum, cells or serum prepared by using 8.1 reuse, or: high pressure of triglyceride or metabolic syndrome. ^ 3 White Lipase Degrading Enzymes - The set of the same set of genes is selected from the following set of genes. 29 The Republic of China December 19, 100 (a) Early set of genes C/AA/T 'includes c located in rs343, AA in 必2 and T in rs253; and (b) a single set of gene GTAG 'which contains 〇 located at AF〇5〇163_25〇〇, located at rs269 where T' is located at rs270 and located at In the G; of rs271, the above-mentioned homologous marker is detected by using an oligonucleotide which can specifically hybridize under the condition of height and lattice, and the appearance of the above-mentioned equivalent gene marker is indicated. High blood pressure, high triglyceride or risk of metabolic syndrome. 9. The method of claim 8, wherein the above-mentioned equivalent gene marker is carried out by using DNA prepared by peripheral blood of a patient. Detection.