CN1936017A - Glucagon-like peptide-1 receptor gene pleiomorphism and Type2 diabetes correlation - Google Patents

Abstract

The invention discloses a method to detect 2-type diabetes mellitus susceptibility that includes the following steps: testing the glucagon-like peptide 1 receptor, checking whether it has dissociation comparing to the normal one. It also discloses the corresponding testing kit.

Description

The dependency of Glucagon-like peptide-1 receptor gene polymorphism and diabetes B

Technical field

The present invention relates to biology field.Relate more specifically to people's Glucagon-like peptide-1 acceptor gene (glucagon-like peptide 1 receptor, single nucleotide polymorphism GLP1R) (singlenucleotide polymorphism, SNP) and with the dependency of diabetes B.The invention still further relates to the method and the test kit that detect these SNP.

Background technology

It is the syndrome of being made up of the different pathological physiological change of feature with the abnormal carbohydrate metabolism that diabetes are one group, have bigger heterogeneity between the different patients, and its two kinds of main pathophysiological changes is hypoinsulinism and insulin resistant.

Though carried out the research of some diabetes B genes involveds and polymorphism, before the present invention, do not confirm the report of GLP1R gene pleiomorphism and diabetes B dependency, more there is not report with the diabetes B dependency.

In sum, in view of diabetes have a strong impact on the healthy disease of people, in order to diagnose and treat diabetes B as early as possible, this area presses for seeks the diabetes B tumor susceptibility gene, and method, the test kit of exploitation detection diabetes B, or relevant medicine.

Summary of the invention

Purpose of the present invention just provides the method and the detection kit of a kind of diagnosis (especially early diagnosis) diabetes B.

In a first aspect of the present invention, a kind of method that the diabetes B susceptibility of individuality is diagnosed is provided, it comprises step:

Detect this individual GLP1R gene, transcript and/or albumen, and compare with normal GLP1R gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble diabetes B is higher than normal population.

In another preference, what detected is gene or the transcript of GLP1R, and with normal GLP1R nucleotide sequence comparing difference.

In another preference, described difference is the single nucleotide polymorphism that is selected from down group:

The genotype in 4347 G A and this site is AA;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

In a second aspect of the present invention, provide a kind of test sample whether to have the method for the single nucleotide polymorphism of GLP1R gene, comprise step:

(a) with the GLP1R gene of GLP1R gene-specific primer amplification sample, obtain amplified production; With

(b) detect whether there is the single nucleotide polymorphism that is selected from down group in the amplified production:

The genotype in 4347 G A and this site is AA;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

In another preference, described method also comprises step: the sample supplier carried out the BMI index analysis.

In a third aspect of the present invention, a kind of test kit of complementary detection diabetes B is provided, it comprises the primer and the specification sheets of specific amplification GLP1R gene or transcript.

In another preference, it also contains the reagent that is selected from down group described test kit:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

In another preference, described sudden change is the single nucleotide polymorphism that is selected from down group:

4347 G A;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

In another preference, described primer comprises the primer of sequence shown in SEQ ID NO:2,3 and 4.

In a fourth aspect of the present invention, a kind of GLP1R gene and proteic purposes are provided, they are used to prepare the reagent of complementary detection diabetes B.

Description of drawings

Fig. 1 has shown the SNP image in the rs2268657 site of GLP1R.

Embodiment

The inventor finds first and has proved that through research for many years GLP1R gene and diabetes B are closely related.The inventor has carried out order-checking and snp analysis to a plurality of zones in the GLP1R gene, wherein the association study result shows, compare frequency with the regular reorganization of BMI＜25 there were significant differences (OR=1.939 in the frequency of genotypes AA of GLP1R rs2268657 site (corresponding to 4347 among the SEQ ID NO:1) the normal people, P=0.022), compare no significant difference with the obese diabetes patient group of BMI＞28.This prompting, GLP1R gene polymorphism sites rs2268657 and hypoinsulinism are that the diabetes B of leading is relevant, can be used for the auxiliary detection diabetes B.Finished the present invention on this basis.

Particularly, the inventor chooses diabetes B patient 360 examples and normal control 313 examples of affinity-less relation, wherein the diabetic subject is divided into fat group 192 people (BMI＞28, and only treat with oral antidiabetic drug) and regular 168 people (BMI＜25 of recombinating, and insulinize), adopt allele specific oligonucleotide PCR in real time (allele-specific real-time PCR), rs2268657 carries out gene type to the GLP1R gene locus, and studies the dependency of this site and diabetes B by correlation analysis.

The result shows that GLP1R gene rs2268657 loci gene type frequency AA, AG, GG in the contrast crowd are respectively: 27,140,146; AA, AG, GG are respectively in the normal type diabetic groups: 26,63,79, in the obese diabetes group, AA, AG, GG are respectively: 21,96,75 frequency of genotypes AA are compared frequency there were significant differences (OR=1.939 in the regular reorganization of normal people and BMI＜25, P=0.022), compare no significant difference with the obese diabetes patient group of BMI＞28.This result of study prompting GLP1R gene polymorphism sites rs2268657 and hypoinsulinism are that the diabetes B of leading is relevant.

As used herein, term " GLP1R " and " Glucagon-like peptide-1 acceptor " are used interchangeably, and all refer to the protein molecular or the nucleic acid molecule of Glucagon-like peptide-1 acceptor.

The detailed sequence of GLP1R gene can be referring to accession number Hs.389103Nucleotide sequence (can referring to network address http://www.ncbi.nlm.nih.gov/), total length is 38903bp (SEQ ID NO:1).The rs2268657 site is corresponding to the 4347th among the SEQ ID NO:1.

Based on new discovery of the present invention, GLP1R albumen or polypeptide have many-sided new purposes.These purposes include, but is not limited to: the disease that complementary detection is relevant with GLP1R (as diabetes B), or the material of screening promotion GLP1R protein function, and as antibody, polypeptide or other part.The peptide molecule that can stimulate people GLP1R protein function that can be used for seeking therapeutic value with the recombinant human GLP1R protein screening peptide library of expressing.

On the other hand, the present invention also comprises people GLP1R DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people GLP1R gene product or fragment.Preferably, refer to that those can combine with people GLP1R gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of people GLP1R, comprise that also those do not influence the antibody of people GLP1R protein function.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people GLP1R gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human GLP1R albumen or its have antigenic segmental cell and can be used to immune animal and produce antibody.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund's adjuvant etc.

Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Antibody of the present invention comprises the antibody that can block people GLP1R protein function and the antibody that does not influence people GLP1R protein function.Each antibody-like of the present invention can utilize the fragment or the functional zone of people GLP1R gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people GLP1R gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

The proteic antibody of anti-people GLP1R can be used in the immunohistochemistry technology, detects the people GLP1R albumen in the biopsy specimen.A kind of preferred anti-GLP1R antibody is the antibody of the normal GLP1R of nonrecognition GLP1R but identification suddenlys change, perhaps discerns normal GLP1R but the antibody of nonrecognition sudden change GLP1R.Utilize this antibody to the proteic specificity difference of normal and unusual GLP1R, the diabetes B susceptibility that can carry out protein level easily detects.

Utilize GLP1R albumen of the present invention,, can filter out with GLP1R albumen interactional material takes place, as inhibitor, agonist or antagonist etc. by various conventional screening methods.

GLP1R albumen of the present invention and antibody thereof, inhibitor, agonist, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intravenously or subcutaneous administration.

In pharmaceutical composition of the present invention, contain the activeconstituents relevant (as GLP1R albumen or its antibody, inhibitor, agonist, antagonist) and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount with GLP1R albumen.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 0.1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that the GLP1R albumen of safe and effective amount or its antagonist, agonist are applied to Mammals, wherein this safe and effective amount is usually at least about 0.1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 0.1 microgram/kg body weight-Yue 100 micrograms/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The invention still further relates to the diagnostic testing process of quantitative and detection and localization people GLP1R protein level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.

Whether having the proteic method of GLP1R in a kind of test sample is to utilize the proteic specific antibody of GLP1R to detect, and it comprises: sample is contacted with the GLP1R protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample GLP1R albumen.

The proteic polynucleotide of GLP1R can be used for the diagnosis and the treatment of GLP1R protein related diseases.Aspect diagnosis, the proteic polynucleotide of GLP1R can be used for detecting the proteic expression of GLP1R GLP1R abnormal exprssion whether or under morbid state.Can be used for the hybridization of biopsy specimen to judge the proteic abnormal expression of GLP1R as the GLP1R dna sequence dna.Hybridization technique comprises the Southern blotting, Northern blotting, in situ hybridization etc.These technological methods all are disclosed mature technologies, and relevant test kit all can obtain from commercial channels.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of GLP1R albumen and also can detect the proteic transcription product of GLP1R.

The sudden change that detects the GLP1R gene also can be used for diagnosing diabetes B.Detection can be at cDNA, also can be at genomic dna.The form of GLP1R protein mutation comprises that the point mutation compared with normal wild type GLP1R dna sequence dna, transposition, disappearance, reorganization and other are any unusual etc.It is the SNP of GLP1R that one class is preferably suddenlyd change, especially the SNP in rs2268657 site.Available existing technology such as Southern blotting, dna sequence analysis, PCR and in situ hybridization detect sudden change.In addition, sudden change might influence proteic expression, therefore can judge indirectly that with Northern blotting, Western blotting gene has or not sudden change.

Major advantage of the present invention is:

The used experiment confirm first change of GLP1R and the closely related property of diabetes B, and then the method and the test kit of complementary diagnosis diabetes B (or susceptibility) are provided.The present invention can provide very valuable complementary reference index for clinical diagnosis (especially early diagnosis) diabetes B, thereby benefits diagnosis morning, prevention early, the morning that help realizing diabetes B.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The inventor is in the small sample scope, order-checking and snp analysis have been carried out in a plurality of zones in the GLP1R gene, wherein the association study PRELIMINARY RESULTS shows, is a pleomorphism site on the intron in GLP1R the 4347th site, and there are dependency in this site rs2268657 and diabetes B.For further clearly whether this site is relevant with the Chinese han population diabetes B, selected the detection crowd again at random, carry out the correlation research analysis.

One, object and method

1) research object:

The full-fledged research object is all from CHINESE REGION, to each other consanguinity-less relation.Normal people totally 316 people wherein, diabetes B patient (meeting WHO Case definition in 1999) (Report of a WHO consultation.Definition, diagnosis and classification of diabetes mellitus and itscomplications.Geneva:World Health Organization, 1999:52.) totally 366 people, the diabetes B patient is again according to weight index (BMI calculating=body weight (Kg)/height ²(M ²)) being divided into fat group and regular reorganization, fat group is totally 197 people, BMI＞28m ²/ kg, and only treat with oral antidiabetic drug, the regular reorganization is totally 169 people, BMI＜25, and insulinize, all capable 75g oral glucose tolerance test (the BECKMAN LX20 biochemical instruments of all selected objects, glucose oxidase electrode method), wherein normal people fasting plasma glucose＜6.1mmol/L and postprandial blood sugar＜7.8mmol/L, diabetic subject fasting plasma glucose＞7.8mmol/L and/or postprandial blood sugar＞11.1mmol/L, and get rid of the diabetes of type 1 diabetes, MODY and matrilinear inheritance according to personal history and family history.

The physique measuring method: experimenter's slippers, only wear underwear, measure height and body weight.

The height measuring method---the measured is barefoot, heel closes up, and toe is separated into 60 ° of angles, and upper limbs is sagging naturally, trunk is straight and upright naturally, keep in touch with metope between foot root, sacral region and two omoplates, head is ajusted, but needn't be near metope, two the place aheads at ordinary times, keep being in same sea line because of the eye socket lower edge on the tragus, the measurer stands in the measured side, measures the height of crown horizontal plane apart from ground.

Measured body weight method---measured is lacking clothing as far as possible and is standing gently in scale central authorities, reading when treating that registration is stablized.

Control group		Diabetic groups
				BMI＜25, insulinize	Only treat with oral antidiabetic drug BMI＞28
		Age-sex (man/woman) weight index Kg/M 2Fasting plasma glucose mmol/L postprandial blood sugar mmol/L	58.4±10.96 92/224 21.8±2.10 4.47±0.59 4.71±1.29			64.1±10.20 72/97 21.6±2.20 11.7±4.20 9.32±3.40	62.6+9.50 67/130 30.3+3.20 17.4±6.10 12.86±8.80

2. gene type principle

The single-minded PCR of this experiment employing allelotrope combines with fluorescent real time PCR and carries out gene type, its ultimate principle is: at two primers of 5 ' upstream design of DNA polymorphic site, make 3 ' of primer hold last base just in time to be on the detected polymorphic position, wherein 3 ' base of a primer and a kind of allelotrope are complementary, and another primer then mates with another kind of allelotrope.Use the archaeal dna polymerase of 3 ' terminal specific identification, this enzyme can be discerned the Nucleotide of primer 3 ' end specifically, have only Nucleotide and the template of working as this position to mate fully, reaction just can continue, as do not match, reaction efficiency then reduces greatly, in reaction system, add the Sybergreen fluorescence dye in addition, this fluorescence dye can be tied under the activation that is incorporated in laser with double-stranded DNA and send fluorescence, the amount of its intensity and double-stranded DNA is proportional, fluorescent signal is detected and record after each loop ends, just obtains two kinds of right fluorescence curves of different primers of each sample after whole PCR reaction finishes.Pairing circulation thresholding (Ct) judges that this sample is any genotype when arriving the thresholding of setting by the fluorescent signal in each reaction tubes in the more different primer PCR processes.Circulation thresholding as two kinds of primers differs (Δ Ct) more than 5, then is homozygote, and the circulation thresholding differs and is heterozygote below 1, and the sample repeated experiments between 1 and 5 is to clear and definite somatotype.

3. method

1) DNA extracting:

All objects are taken out 3 milliliters of peripheric venous bloods, and phenol/chloroform method extracting genomic dna routinely.

A.3ml fresh blood adds mixing in the test tube of 0.8ml acid-citrate-dextrose solution (ACD), in time extracting DNA after extracting DNA or-70 ℃ of frozen the treating;

B. anticoagulation 3ml moves into (frozen blood melts the back operation in room-temperature water bath) in the 50ml plastic centrifuge tube, adds the haemolysis reagent of 10ml precooling, fully mixing;

C. ice bath is 10 minutes, and 4 ℃ of following 5000rpm 10 minutes abandon supernatant, and precipitation adds 10ml haemolysis reagent, repeats this step once;

D. add 4ml nuclear suspension, mixing gently in the white precipitate;

E. the 10%SDS that adds 50 ℃ of preheatings of 100ul adds 40ul Proteinase K (20mg/ml) again, and making final concentration is 200ug/ml, in 45 ℃ of 100 time/minute shaken overnight, fully digestion gently;

F. add 4ml TE in above-mentioned digestion solution, this step can be selected to add TE or do not add TE according to actual digestion situation;

G. add equal-volume Tris balance phenol (PH8.0) again in above-mentioned centrifuge tube, be mixed into milky white shape gently, centrifugal 10 minutes of 4 ℃ of following 6000-7000rpm move into another cleaning 50ml centrifuge tube with supernatant liquor;

H. repeating step g;

I. add the freshly prepared phenol-chloroform-primary isoamyl alcohol of equal-volume (25: 24: 1) mixed solution in above-mentioned supernatant liquor, mixing is centrifugal the same gently.Supernatant liquor is moved into another cleaning 50ml centrifuge tube;

J. the dehydrated alcohol that adds-20 ℃ of precoolings of the sodium acetate soln (3mol/L) of 1/10 volume and 2 times of volumes in this supernatant liquor turns upside down gently to flocks and separates out;

K. after flocks is separated out 30-60 minute, choose precipitation, put into the 1.5ml Eppendorf pipe that is added with dehydrated alcohol in advance with aseptic Tip (high pressure is crossed), centrifugal 20 minutes of 13000rpm, abandon ethanol, DNA places seasoning in the unlimited pipe, until the volatilization of visible trace ethanol totally.Adding 600ulTE dissolves DNA;

L. measure the absorbancy of DNA sample, A at 260nm and 280nm place ₂₆₀/ A ₂₈₀Ratio illustrates that about 1.8 the extracting quality is better, and is frozen stand-by with measuring good DNA numbering, packing ,-20 ℃.

2) PCR reaction

Utilize the special software SNP PRIMER DESIGN of Roche Holding Ag to design, principle of design is with the 3 ' end of mutational site as upstream primer, designs two special primers, consensus primer of downstream design, between the fragment length 35-60bp, primer length 15-23bp.Design and synthetic following primer:

Upstream primer 1:CTTGTTGGATCTGTGCAGTT; (SEQ ID NO:2)

Upstream primer 2:TTGTTGGATCTGTGCAGTC; (SEQ ID NO:3)

Downstream consensus primer: CCAGTTCTGCCGTCCATAAAATG; (SEQ ID NO:4)

Product sequence: CTTGTTGGATCTGTGCAGT[T/C] TGAGAAGAATGATATGCCCATTTTATGGACGGCAGAACTGG.Use the fluorescence real-time quantitative PCR instrument to carry out the PCR reaction, response procedures is as follows: 95 ℃ 10 minutes, preheating, 95 ℃ 30 seconds, 58 ℃ 30 seconds, totally 45 circulations during each loop ends, detect fluorescent signal automatically.

3) reaction system

Calculate with every hole 50ul reaction system, 96 orifice plates can be made 44 patient's sample, add following mixture in every hole respectively

Damping fluid (0.05M tricine, 0.05M potassium acetate, 3mM magnesium acetate)	10ul
		dNTP	1ul
The Gold archaeal dna polymerase	0.1ul
		20×Sybgreen	0.5ul
DMSO	2ul
		Glycerine	1.24ul
H 2O	32.16ul
		Downstream consensus primer (concentration is 20ng/ml)	1ul

In two pipes, add special primer 1 and primer 2 respectively again, 1ul (concentration is 20ng/ml), the vibration mixing is got the 96 orifice plate (AXYGEN that are exclusively used in fluorescent PCR ^), every hole adds dna profiling 1ul (concentration 20ng/ul), and adjacent sample that two rows add is the same, added mixed system is added every hole adds 49ul in the entering plate, and the interlacing adding contains the mixed system of primer 1 and primer 2.Use fluorescent PCR instrument ABI ^PRISM 7000 (ABIBiosystem).

4) the PCR in real time result judges:

The situation difference of different amplified alleles, (Ct1, difference DELTA Ct Ct2) judges genotype to the Ct value by two Ct curves relatively and the intersection point of the threshold value that sets.Δ Ct=Ct1-Ct2 is if Δ Ct＞5 then are that A is allelic for homozygote; Δ Ct＜-5 item are the allelic homozygote of B; | Δ Ct|＜1 then is a heterozygote (Fig. 1).

5) reagent and instrument:

DNA extraction agent: Proteinase K (Merk company)

The ACD antithrombotics: citric acid 0.48 gram+Trisodium Citrate 1.32 gram, add distilled water to 94 milliliter, behind the autoclaving, add 6 milliliter of 25% glucose injection;

Haemolysis reagent: 1 milliliter+1M of sucrose 109.54 gram+1M Tris-HCl MgCl ₂10 milliliters of 5 milliliters+TritonX-100 add distilled water to 1000 milliliter, mixing;

Nuclear suspension: 24 milliliters of 7.5 milliliters+0.5M of 5M NaCl EDTA add distilled water to 500 milliliter; 80 milliliters of 10%SDS:SDS10 gram+distilled waters, the HCl accent PH to 7.2 with 1M is settled to 100 milliliters;

TE damping fluid: 0.5 milliliter of 2.5 milliliters+0.5M of 1M Tris-HCl EDTA adds distilled water to 250 milliliter;

10000 * fluorescence dye SyberGreen is available from genome company;

5 * fluorescent PCR damping fluid: Tricine8.96 gram+potassium acetate 4.907 gram+magnesium acetates 0.6434 gram, add distilled water to 1000 milliliter, transfer PH to 7.4-7.6 (AMRESCO company) with the NaOH of 1M;

DMSO(SIGMA)；

Allele specific oligonucleotide archaeal dna polymerase Delta Z05 Gold DNA Polymerase (ROCHE);

Real-time quantitative PCR adopts ABI  PRISM 7000 (ABI Biosystem);

BioPhotometer spectrophotometer (Eppendorf);

Cyberscan2500 PH counts (EUTECH);

5818 R refrigerated centrifuges (Eppendorf).

6) statistical analysis:

Adopt SPSS analysis software (SPSS Inc) to carry out statistical study, Pearson X is adopted in the test of significance of gene frequency group difference ²Check, the Logistic regression analysis is adopted in genotype and disease-related analysis, and to represent than (OR) than number.X is used in the Hardy-Weinberg equilibrium analysis ²Check, and use the FINETTI program (http://ihg.gsf.de/cgi-bin/hw/hwal.pl)Carry out statistical study.

Two. the result

GLP1R gene polymorphism sites rs2268657 allelotrope and genotype frequency distribute and see Table 2, meet the Hardy-Weinberg balance through the check genotype frequency.Gene frequency distribution (A/G) does not have significant difference in each group, but the frequency distribution of frequency of genotypes AA/AG+AA is variant.

The AA genotype in the normal type diabetic groups apparently higher than normal control group (X ²=5.23, P=0.022 OR=1.939) (sees Table 3), and compares no significant difference with the obese diabetic group.Show that allelotrope A is relevant with the normal diabetes B morbidity of body weight under the recessive inheritance pattern, irrelevant with the diabetes B morbidity of obesity.

Allelotrope and genotype frequency distribute between the different subgroups of table 2. control group and diabetes

Group	Genotype allelotrope
						AA(％)	AG(％)	GG(％)	A(％)	G(％)
	The fat group of control group regular reorganization	27(8) 26(15) 21(11)	140(45) 63(38) 96(50)	146(47) 79(47) 75(39)	194(31) 115(34) 138(36)						432(69) 221(66) 246(64)

Equipotential gene A and diabetes correlation analysis under the different hereditary patterns of table 3.

Group	Dominant inheritance (GG/AG+AA)				Recessive inheritance (AA/AG+GG)
									OR	95％CI	X 2	P	OR	95％CI	X 2	P
	Control group/normal type diabetic groups control group/obese diabetic group	0.985 1.364	0.676-1.434 0.947-1.965	0.01 2.78	0.937 0.095	1.939 1.301	1.091-3.446 0.713-2.372	5.23 0.74									0.022 0.390

Results suggest, there are dependency in the SNP and the diabetes B in GLP1R gene rs2268657 site.Allelotrope A is relevant with the normal diabetes B morbidity of body weight under the recessive inheritance pattern, and is irrelevant with the diabetes B morbidity of obesity.

Three. discuss

Just find the insulin secretion effect that Glucagon-like peptide-1 (Glucagon-like peptide-1GLP1) has stimulates glucose induction as far back as Holst in 1986 etc., again find that GLP1 can significantly improve the susceptibility of B cell to glucose, improves the B-cell reactivity of NOD mouse thereafter.The effect that studies show that GLP1 is to mediate by the special acceptor Glucagon-like peptide-1 (GLP1R) on the B cell film, with insulin secretion important relation (Chen Yue is arranged, Chen Jialun. Glucagon-like peptide-1 and to the regulation and control .Chinese Journal of Diabetes of glucose level, October 1999, Vol 7, No5:290-291).

Oral glucose can cause plasma insulin level raising by a larger margin than the glucose intravenous injection of Isodose, this sugared incretin effect is because some gastrointestinal hormone ` is released into blood, act on B cell, strengthen due to the insulin secretion process of glucose induction.These hormones are collectively referred to as incretin (incretin), and wherein Gastric inhibitory polypeptide (GIP) and Glucagon-like peptide-1 (GLP-1) are considered to most important two kinds and regulate relevant incretin with sugar.Studies show that GIP and GLP-1 acting in conjunction just can obtain whole incretin effects, wherein the effect of GIP accounts for 30%, and GLP-1 accounts for 70%.Therefore, GLP-1 is hormone medium [Fehmann HC important in the entero-insular axis, Goke R, and Goke B.At the cuttingedge:Glucagon-like peptide-1 (7-37)/(7-36) amide is a new incretin.Mol Cell Endocrinol, 1992,85:39; Goke R, Fehmann HC, Linn T, etal.Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide and antagonist at the glucagon-like peptide 1-(7-36)-amidereceptor of insulin-secreting beta-cells.J Biol Chem, 1993,268:19650.].And the effect of GLP-1 is to mediate by the special acceptor GLP1R on the B cell film.People GLP1R gene is positioned on No. 6 the short arm of a chromosome, 463 the amino acid whose protein of encoding, contain seven disconnected for striding diaphragm, belong to heterotrimer G2 protein linked receptor superfamily (HeterotrimericG-protein coupled receptor superfamily), this receptor is at pancreas islet, lung, there is high-level mRNA to express in hypothalamus and the stomach, GLP-1 combines with B cell surface GLP1R, cAMP and calcium ion level in can regulating cell, finally played insulin secretion effect [the Leech CA that strengthens glucose induction, Holz GG, Habener JF.Signal transduction of PACAP and GLP-1 in pancreatic betacells.Ann N Y Acad Sci, 1996, Dec 26; 805:81-92; Discussion 92-3; HolzGG, Leech CA, Habener JF.Activation of a cAMP-regulation Ca2+-signaling pathway in pancreatic beta-cell by the insulinotropic hormoneglucagons-like pep tide-1.J Biol Chem, 1995,270:17749; Perfetti R, Merkel P, Glucagon-like peptide-1:a major regulator of pancreatic β cell function, Eur J Endocrinol, 2000,143:717-25], the GLP1R gene knockout mice impaired glucose tolerance can occur and blood insulin levels reduces, particularly the post-stimulatory Regular Insulin of glucose discharges and reduces (Scrocchi L, Brown T, Maclusky N, et al., Glucose intolerance butnormalsatiety in mice with a null mutation in the glucagon-like peptide1 receptor gene.Nat Med, 1996,2:1254-8).

Above-mentioned studies show that, GLP1R and B cell insulin secretion function have close getting in touch.The present invention studied in the GLP1R intron pleomorphism site rs2268657 and and the Chinese han population diabetes B between dependency.Because it is the syndrome of being made up of the different pathological physiological change of feature with the abnormal carbohydrate metabolism that diabetes are one group, has bigger heterogeneity between the different patients, and its two kinds of main pathophysiological changes are hypoinsulinism and insulin resistant, carry out layering if therefore can change the diabetic population of forming to these different pathological, make it become the single relatively disease crowd of the cause of disease, then will help the detector efficiency of tumor susceptibility gene.Usually insulin secretion and insulin sensitivity can be assessed by clamp test or HOMA index, but clamp is tested owing to its complicated operation, is cost dearly, can't be used for large-scale clinical trial, but though and the insulin secretion situation and the insulin sensitivity of HOMA index coarse evaluation individuality, this index can be subjected to the influence of factors such as the sensitivity of the course of disease, pharmacological agent and detection method and stability.

This research is attempted using simpler, metastable index is analyzed diabetic population simultaneously, be divided into regular reorganization (BMI＜25) and fat group (BMI＞28) by weight index, wherein the regular reorganization is the patient of insulinize, and fat group is then only treated with oral antidiabetic drug.Existing data shows, the former pathological change is many based on hypoinsulinism, and the latter's pathological change many be feature with the insulin resistant, though two kinds of pathological changes can exist simultaneously.The present invention studies have shown that the pleomorphism site rs2268657 of GLP1R is relevant with the diabetic population of normal type and insulinize, and does not have significant correlationship with diabetic population fat and that only treat with oral antidiabetic drug.From present research, the effect of GLP1R is main relevant with insulin secretion, so the present invention points out the rs2268657 site may be relevant with the diabetes B that with the hypoinsulinism is feature.

Certainly, because this pleomorphism site is positioned at the intron of gene, therefore whether this polymorphism can directly cause the function of gene or express change, perhaps only is a polymorphism sign that is linkage disequilibrium with real pathogenic pleomorphism site, awaits further to studies confirm that.The present invention also shows simultaneously, by some simple clinical indices, as BMI, pharmacological agent situation etc. patient is carried out preliminary layering, helps to reduce patient's heterogeneity, and improves the detector efficiency of correlation analysis.

Embodiment 2

Diabetes B susceptibility detection kit

Prepare a test kit, it contains:

Title forward primer 1	Sequence (5 ' → 3 ') CTTGTTGGATCTGTGCAGTT	Numbering SEQ ID NO:2	Concentration dry powder 20D
				Forward primer
2	TTGTTGGATCTGTGCAGTC	SEQ ID NO：3	Dry powder 20D
				Reverse primer	CCAGTTCTGCCGTCCATAAAATG	SEQ ID NO：4	Dry powder 20D
The PCR reaction solution	Contain Taq enzyme dNTP magnesium ion PCR reaction buffer

Extract the blood 3ml of 90 arbitrary object to be detected (BMI＜25), use ordinary method (or using specific test kit) from blood, to extract DNA.Press the identical method of embodiment 1, PCR primer in the diabetes B detection kit is diluted to 1 μ mol/ μ l, with the DNA that is extracted is that template is carried out the PCR reaction with the primer that is provided, and carry out fluorescent real time PCR and detect, thereby the genotype at judgement GLP1R gene polymorphism sites rs2268657 place.

Detected result as shown in Figure 1.Wherein, the detected object of frequency of genotypes AA is carried out conventional fasting plasma glucose again and the sugar tolerance detection method detects whether suffer from diabetes.The result shows, genotype is to have 12 to suffer from diabetes Bs in 18 detected objects of AA.This detection can ordinary method fail to detect diabetes in, the reflection testee suffers from the possibility of diabetes.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Endocrine-Metabolic Diseases Inst.

＜120〉dependency of Glucagon-like peptide-1 receptor gene polymorphism and diabetes B

<130>050054

<160>5

<170>PatentIn version 3.1

<210>1

<211>38903

<212>DNA

＜213〉homo sapiens (homo sapiens)

<400>1

atggccggcg cccccggccc gctgcgcctt gcgctgctgc tgctcgggat ggtgggcagg 60

gccggccccc gcccccaggt gagatccagg gaccccgacg acaccggggg aggcgggtgg 120

ctctgcgggc tgcaggcgca gtgtcctggt ggagggcccc gggacttgaa cgaaactccg 180

agaccgctgg cgggggcatc cgaaagcctc ggggggagta gggactggag acttggtgcc 240

cctgggctgg aaggcccagg tgagggcttg gactacagag gattcccccc aaccccagcg 300

aggcgccctc ttcctcgcca cccgggtccc tctgcccggg cacccgctgc cccgcggccg 360

ccctgcgctg acttctgctc cgcttctcct ttgtccccag cactttccca actccttcta 420

aaccgtgtca gcggccctgg cacagcccgg catcctcccg gactccctct gccccacagt 480

ctgacccaga cccctctccc gcagtgcctc cccagacaat ccacctgctc aaagaccctg 540

agaagaccct gggaggagct gagagggcca gggtgcggag acccggctcc tctccctttc 600

tcatcagccc ccagcacagc ctttgattca tggctcctgg ggccctgcgg tggcagcttg 660

tccttcccag tgacctccca gatggaacct accccccgtc ccctaccagc ctctgtagca 720

aacaggcaga aggctcagtg cccccctgga agcagccagg cgcccccact cacccactct 780

gctgacctcc cattctcatc ctgcactgac agcaggcccc aagagaaggc atactgggac 840

acgcaagaac acctgggctc cttggtgcca ccagagagca ggccacaggt ggaccaccgc 900

agcccctctc tcttgccctt gttgcttttc ctgccctggg atcctgggct agggacagtg 960

gtcagctgct tttgaagagg ggagctgtcc aaagatgctg ggacttgctt gagcagagag 1020

gagacataga cttgggtggc ctgtcctggg tcaggaaaca ggttcttcct atcacctccc 1080

actgctaccc ccagacctaa agaactgaga tctgtgggga gtggacctga acgaaccttg 1140

aagtttgtcc cagaaccatt gccttggtga gggcagactc cacctcggcg gaaaccccag 1200

atgtggacag acatacctct cagctaagat gccagcacat ttttcagccc ccagattatt 1260

ttccagacgc atcctctccc tgcccctgtt ccccagccca gctgctgtcc tgagccccag 1320

gagacaacct cccccttcca agacaccttc ctcacagcgc ccccagccag gccttggttt 1380

tcctggtgcg gctgggatct ttcaggggcc agggctgcag acccaagatg tggttcttgc 1440

ccatcgttcc ctcctcttac ggactgggtt gggatagggg tggaggctct gcttcctggt 1500

tccatcctcc tccaggccag gtttacaggt ctggtttgaa tgaagggaag aaagcaaagg 1560

gaagaggaaa gtttctgtga gctgtcttcc cagaggcctt attcccagaa gtgtcaggct 1620

cctgaaccca cactttcctc tgatccgatt ttactcctta gccccaggcc taaggcaaaa 1680

gctgggcatc ctgggggcat aacaggattg gggtggtaga gaaggatagg aggggacagg 1740

tcctagcagg gaggggagct ttgagggggt gctcttgtca tgggaaactg agctgccaca 1800

gagatctgag tcacaggagg tgttaaggat agattgcaac aattatgaca caacatcatg 1860

cgtgcttata agactaaaaa atagtgtatt ttgcattttg ttttctgtct atgaagtcaa 1920

aagggttaga gttactcccc tcatgttgct gatgggggaa tgaaagctca gaaaaagagg 1980

gtcccctgta taaagtagcc cagcagttca ggagcaggga agagaaaggg gtgaagggct 2040

gggctcttga cctaacacct cctactcaga cgggactgaa gggagacctc tgagagtgag 2100

ctgctcactg tcaggtctcg tgaggctgga aacagcatcc ctgcaggtag ggggatggct 2160

gagttgactt ctgaagaagg tgccctaggc tactctttca ttgctgctgg gaaggctcat 2220

tcccctaccc tggtccttac ctctccaata ccctggtgag ggagcgggcc tactgaggag 2280

acaggcaagc ctttgacact caaagttaaa atcagacaca actggattca aatatagggt 2340

ctgctattag ctggtgatct ggagttggac acttggttat ccccaataca tgtccttatc 2400

tgtaaataag gatcataagc tctatcacat agggttatgg aggggattga gacactggcc 2460

cctgaaatgg caactttagt cactttagtc aaatggcaga actgacccat ttgtccttgg 2520

ggatccagaa gtgttttcca gggctgcagg atagtatgag agaggggctg ttggagaccc 2580

tgggtctctg tactctagtg aatggcaggt ggagtccagg gctgagcctt gatcagtgcg 2640

gacattgttc taagctgggt ttcctcctgg acaggaccca ggctgggggc tggggcagga 2700

gcaaccaaga aaccacatcc tgccctcgaa tgccttgcac caaggcaaaa caggcttcct 2760

cagctgcagc ctgcccctag gaaaattcct atgtgtgtgt ggtggtggtg gtggttggag 2820

gaggggacag agatacagac aggtagtctg agacagacca gggggagaag ggagtaggct 2880

atatgaaccc ccagttccca ggtatgtgtg agtgaccctg tgtgtaacag tctgtgtggg 2940

aatgagtccc tgtgtgtgca acatctcagc atgcaactgt acatctgaca gtgtctatgt 3000

ttttgtgacc ctgcatatga tactgtgtct gtgggagggt ctgtgtgtgt gtgtgggggg 3060

gggggcatct tggtgtgtga atgggtgtca actagtgtgt gtctatatgt gcaggagaga 3120

tagtgtgcat gtgagagtgt ctgtgtgttc ctctgtgtgt gtgtgttggg gacagtgtct 3180

cagcatgcga ctgtgtacct ggcagtctgt gagtgattgt gtgtggtggg agagagtctg 3240

tgtcagtgtc tcagtattgg gaatgtgctt gggggcgtgt gtgatgatgc gtgtgaccgt 3300

gtgtccacgt gggggttttt gtgactgtaa gaacatgtgt gtagtattcc ttttcctgtc 3360

ctttcgttca gatatctaaa gcccttaggg aaaaaaaatg gattgtaagc tctgagcttt 3420

tgaaaaaaaa gaaagaaaga aagaaaaaca aggggcggtc aaagagacag gggtctgttg 3480

atggaagggg tgatttgact tctgggaggt gacacagagg aaggaggttg gggtcctggg 3540

aggagatgcc agggaacctg aagggctgga cttagttggg ggactccaga aatttgagtt 3600

ggaatccctc cttcctcctt ctgagaggcc ttcctgcatg tctcccacct ctgtgccacc 3660

tccttcccgt caccaaaatg gtccccagag aaagaaagaa tctgatacac ttattcgcac 3720

tgagaaatag gagtgccttt cagattggca gccagtgttt ggagagggag ccccttcctg 3780

gggtgttgga aagcccacca ccccgggggc ttccactcca cctgcttcct tctctgctgt 3840

ccttccttcc aacgctgcct gcttgtacag ggcttgagaa gtcacaaaga ttttcacctg 3900

ctttatcttg ttggatctgt gcagtttgag aagaatgata tgcccatttt atggacggca 3960

gaactggcaa ctgatgagga aactgaggtc ctaaaagata aatcgtgtcc cacagtcctg 4020

cattaacaga gctgggattt agaccaagaa cttataaatc gctgtccagt gctccttcct 4080

gtagaccaca ccactccctc ctggtgtcct ctttcctgtc tcctgcttcc tccaaaaact 4140

cctgaatctg agggatgccc cttccttctt gtaacctcca acccaggtgt cgtctccacg 4200

tgccttcatc cacctcagag agatcccatg aagcctgtca agtcttgatt ctagaaactg 4260

ccatcctgcc cttcccatgg ccctttagac cccctctgac cgctgctttg ctctcctggt 4320

cagctctgct gccttgtgag ggacagggac atctggattg catccttccc gctacccaga 4380

acccacagcc ccaccttgtc aggcttgcag cagggactta tgttgtggga ggatgaaatg 4440

gaggagtggg ggacactggt gggcagggcc ttgcccctcc tcacagggca tggggtccaa 4500

agcttcctcc ctgcttcact ttgccattgg ctgtggttac tgctggaaga tctcacaaat 4560

taagcagaat gattggtcat tggacatgaa catatattca tttcctcttt ctgggaatat 4620

gttgatccta gaagtgagga aggggtgggg ccccagtccc caggaagcct ggtcactccc 4680

tcccactctg ccctttccac taaacagtag aaatcatggg gattgctgcc attgactgag 4740

ccttctccat gtgcccacca tcctaagcat taactcagtc cccacagcca tcccatgggg 4800

gaggggctgc tgttcccttt tgcacaagtg taagctgagc ctaaaacata caactcttgc 4860

actcggttgc aaacctaata tctgggtgac ccaggtccag gcccagatct gagcccaagg 4920

ccagggttgt tcagcctcca ctatgttacc tgggcaccca ggccctatgg cactgggtgg 4980

ccctagcctc atttactcag tctttcccca gaccagaacc tcccatctgt cccagggccc 5040

accaccactc tctccctgcc ccaaaaaatc ctcctgataa cagtccacag ctctcctctg 5100

ctctccggag cactgatgtc cccacctttg taccttttcc ctgctgtgcc ctcctgctga 5160

agtgcccgcc ctcctccttg ctagcccagt ctgcctccaa cccccttcac cccagcccca 5220

cctcacagtc cccaatccct caggcctctt gagtctgcac tgaaccctgc cccaaccact 5280

ccctgaacac tttcacccac tgccttccct gttatgtcac ttgtcctgta aagcattgac 5340

ccccgagtag cctgaagact ctctcaggca gggctcctgc agtcaccttt atactccctg 5400

cattcccctc cacccacgac cacccagaac ctggcacagt tatggtttct gtaaatacga 5460

ggcgtgggag aggccagaag tctgggtagt ttgcactgtg tgtgcctgca ggggtgggat 5520

gaaggctcga aggtatttag aagccctgat ctggtgtagg gaggggacaa gatactctta 5580

ctgtcaggaa tccctcacct aagggggagc cacagcccca tcctcaggaa gctccagtct 5640

gaggggagac acagccccgt cctcagggag ccccagtctg aatatagttt tatgtccaca 5700

gctctgctca gagtaggtga cagcgctgcc ctggggtgca ggaggccaga ttggccttca 5760

tggcgctgtc gttaacccct ccccccgcct ctgggaagaa ctggcttcca tacctctctg 5820

cctgccccag acacaccttc tgccccagtt cccagcagtg accttgtgtg tggtctgctt 5880

tcattctaga aacagaaacc aagggggatt tctccgtctc tctgccctcc acccccagca 5940

gctgctgcat ttaggggcag catggaagag ccctttaccc ggagaagagg gaagaagtga 6000

gaagggaaac ggggcggtgg gggttgatcc accaggactt gctctgatga aaactgtgcc 6060

acctcctcag ctgtggctaa tatcatggac aacacctttg cagctgttat gtttgatttc 6120

ttgttctttt tttccccaag caaatacagt tgacctttgc cctcagaaga gctctgtcct 6180

tcagggtagc ccctgccgcc ttggcacact tatgccaagg atgctgctgg ggcttaaagt 6240

gagctgggcc cttccatggg gcctccgagg cagcaagggg tgagcaaggc aacccaagtc 6300

tctcagggag gtgtgtgata agccagctag aacccgagac tggatccagc atgtgcccag 6360

cacgctacaa ggcaaggggg caagcttcta aaaagaagct attttaattt cagcacacgt 6420

ggacattttt gtgatttcaa aaagcaaaat gttgttttaa attatgtatg ggtcctacat 6480

tttatacatg gggctcttgg gtcatttcag catctgcagc ctctgaagga cgaatattta 6540

taatttttaa ggctgttcag gaaaaaacta aaaagaacga caaccccaag acatagctga 6600

aggaagtgca caaaaacact cagaaggaat ggtggattct cagtgggatg ggtctgtttg 6660

ggtctgtgtg ctgaaaagcc atttcggtga cctcactgga agtggaggct cagctctccc 6720

tactcacagg tgagagggat ccctcccatt gcacgtgtga cttcagtgtt ctggaacctc 6780

cctgacccct tcacctcatg tcctcaggac tcttcctttg ggtggataga agctgggttg 6840

aatattagat ttggttgctg tgtcagaggg gagcacgggg agggggtggg gggtgctgtg 6900

ttgagtgcat ctgtaagtga gtgcgttttg gaggaggaat gctaatggca gacatcccag 6960

ctctttgtag ccctgaacgc cactactctc cccggccccc gcacaccttg cgatattcta 7020

ccatggtcac tgatctgaag gactccagat tctagcctta actctgcttc ctgggggggc 7080

tgtgcaccct tgattcagtt cctctcccac tctggcctgt gtgtcttctt ctagaatggt 7140

tgaaagtgca agaagccctc aggcaggctg actaggcgcc agaaggagct tagagtcctg 7200

ggccctcaga aagtctctct cctaagtgag atcagagatc acaaatgaga cacacacgct 7260

cctttcctcc ttcacccacc cacctcccca aaagcatcaa tttagcagac atttgctggg 7320

tgctcgtgac acatcaagct atgcaccctg ggaggaatga agatgtcaaa ccatccacct 7380

ggggcccctg ggtggcagtg acttattctc actctctttc agaggtggct gatgcctggc 7440

agaatttggg gctttgaggt ttcatggaga ttgagaaacc aagtgtgtta gggggtgagg 7500

gggcaggaga ggggctggct gaacccacag gcctcccata tatgccctcc ccccagggtg 7560

ccactgtgtc cctctgggag acggtgcaga aatggcgaga ataccgacgc cagtgccagc 7620

gctccctgac tgaggatcca cctcctgcca caggtgagtc catgtaggct ccccaccttt 7680

agtgctcccc acccaaccaa cttctggagg actttgatga actcaatgtc catggctggg 7740

agccatttgc ctctatagga tgccacccct gccctctgcc ttcatctcat tgtccaagct 7800

actccttcac ctggatacct tcctttctgc cgaccaaaat cttccttttc ttcaaagtcc 7860

gcggaagcac tgcctcctcc agaaaatctt ccccaggact tctgccatca tttggccctt 7920

actcgtttgc tgtagtctgc tttggggttg ctgtcttatt tctgtcttag ctgtcttatc 7980

tgtgtgtcct tccctgcacc gccggccagc cccccagcta gagcctctca gagcaggcac 8040

ccggcctttc catttttgtc ctcagcacct cccagcctgt gcatggactg agggctccac 8100

aaacatattt ataggaataa atgtttcagg gagttcacaa ggtggtttaa agagcacggg 8160

attggagccc gctgggccta ggttgaaatc ctgactgtaa ttataacacg tgacctaaag 8220

aaggttatct taagtgagat cagaaggaag gtaagctaac atttccctct aatctgtgaa 8280

gtgcaattta agaataggcc catagggatt tttcattgag cacgtactat gttccattat 8340

ctatgtagca tatagtcttg atcccacaaa accctatggg gctggaaata ctcctgtttt 8400

acagatcagg aaacagacat taagttactt gctcaaggtc ctgccactga taagtggcaa 8460

gtgcccctgc ttggctcttg ctgggtgggg aaagtgcttg gcatacagta agcattcagc 8520

agaggttagt ggcatgcagc ctccgaggag gggaggggtc ttggggaagg gagggaaagg 8580

gccatggcca gtcacaagca gggactcaga gactgttctt tctgctccca gacttgttct 8640

gcaaccggac cttcgatgaa tacgcctgct ggccagatgg ggagccaggc tcgttcgtga 8700

atgtcagctg cccctggtac ctgccctggg ccagcagtgg tgagccccct ccccgacctg 8760

gtacctgccc tgggccagca gtggtgaacc ccctccccga cctggtatct gccctgggcc 8820

agcagtggtg agccccctcc ccgacctgat acctgccctg ggccagcagt ggtgagcccc 8880

ctccctgacc tggtacctgc cctgggccag cagtggtgag ccccctccct aacctggtac 8940

ctgccgtggg tcagcagtgg tgagccccct ccctaacctg gtacctgccg tgggtcagca 9000

gtggtgagcc ccctccctga cctggtacct gccctgggtc agcagtggtg agctccctcc 9060

cccaccgtcc cctgtatcga ggaggctgag gtcaaggcgc tgcttccttt cctctgagac 9120

acagccctcc ccacccagaa tgtctttagg cctcctagtg agtggtccgc ctgcctttcc 9180

tggggtgagg ggagcagtca gtagtgataa gtttcatgat ggcctgaggt accacctggg 9240

tctgggcgtg aggccagtgc tggccagtgt gtggggaagg gaaagtaagt gtctgaagag 9300

aaatgcgacc ccttccaatt cagtagtcag gaaaagagct gggtggggac agactcttgc 9360

ggggagggaa gctacccagg gtgtctgctg tgtgtataca tgtatgtatg atatgtgtat 9420

gcaggtggcc catacctggc agtgacattt agctacttcc tcactcttta gaggttcact 9480

gggagaagtg atcagggcat tggtggggtg acgaacccca ccagactcag ccctcctcag 9540

ctcactcctg cctgcaaggg ggaccttgta ctggggaaga tgtgactggg taggtgggaa 9600

ggtcatttca ttgggggtac agagatttgc agaggctgca agaggatccc tgactggagg 9660

gttagttaat gtgccccgct atctatccca gcctcccttg ctgagcccag tttcacccag 9720

gcaccgtgga ggataacagg agacaagcac cctcatggca aggagctccc agtctccata 9780

aggggagaaa agatatttcc ctatcatcat catcatcatc atcatcatca ccatcaccat 9840

catcatcgtc atcatagcca gcatttattg agtacctact gtgtgccaag tcctgcccta 9900

aaagcagggc tttataacta ccctgtggtt atacctgctg tacagatgag gaaactgaag 9960

cacaaaaagt ttaagtcact tgttccaaat gatgtggtca atggcagagc caggatttga 10020

atccaattcg tgctcctatc atcacacctt gaaggggctg gagcccaagc agcaactggc 10080

ccagaaacaa gtgcgggcat gaggttatgc tctggggaag ccaggtgtct tcctggagga 10140

ggtggcctgg tggggcccaa tgtaagcttc accttggcac gtgggactga agtcagatga 10200

ggcccaggct ggtctcctaa aagtactgtc tccttgtcca cactcacagt acactcacct 10260

ctccccaagg agcagggaga gcctgggcct agaaggaggc aggagcctgg gcccatcagt 10320

ggcctgcctt ctccaatact gcctgttgga agcactttcc gtgctacact cttttcatcc 10380

tcacaaccct ctgaggcaag tactctgatt accctcccat tttcagatgg ggaaactaag 10440

gcacagaggt gctcagtgac ttttgcaagc tcacacagcc attagtgggc gacccagaag 10500

cttggtttag agtcagtgtg gctaattgct acaccaaagt cttccttggg ggtgtgtgtg 10560

tgtgcccttg catgtggctc tggcctggag acaggtgagt gatctggatg ccctctgctg 10620

tctgggcaca cctctaggga caggggtgag ctgtctgccc tctcctatga actctgcaat 10680

ctggtggttc tgctgactag ccgtgtaagc aggagcaagt tgccagacct ctctgtgcct 10740

tggttgactc atctgggaga tggtatagag ccaagtgaca tgggtgtcgg gaggaccaca 10800

gggccttgag aaatggtagc tgttaatttt tcccacctcc ctacatacct ccctgacttc 10860

ctgcaataca cacactttct ctgcctccca tagatttgag actggcagct tggccagcgt 10920

ctgctgggcc tctagacccc agggagccag gcgtaggaca gggcccatca ctcagcacta 10980

agccaccccc agctctgcct gccgcttgca gctccatcca tcacctttaa ttttatttga 11040

gcaggaaata ggtcctggtg gtgcccgttt atgagcaaac atggttttat tgctttctcc 11100

atgaagataa cggccactgc acaggccccc tgccagcctt tccccaaccc cgtccccctg 11160

ccactcacca ggctaattta ctcccttttt tgacacttgg aatcaccgac tctgctgccc 11220

tctcctcact ccctgagtta cggatgctca gagacaacta ccacaggctc aagctgcgta 11280

attgctgcct ctggcctgtc ctggagcttt ccttggcaaa ggttggcagc ggaggcttcc 11340

ctgaccccca cccaggtgtg tgggagcagt tctgaagaag gggacagttc tggagctttc 11400

ttgggctgat caagcgagta gcctgtggag aaacgtcaca gtgtgccctg gtgtcaagga 11460

gaaggaaggg agttatctca ggcagggtcc tggcagaaaa catgctcagt tggcaatttg 11520

gagagaattt aatgttgggg aggggtcatt ttcagaggct ggcaaggtaa agggaccaat 11580

gaggcatata aagcaccagg gactacaaca atggaaagct tgagcacccc tagacctgag 11640

aaggaaggga caagaatggg gctggggggt ccccatgaca gctgggctac aatggagaag 11700

ccacccagca gggctgtggc cacagagggg acaaagtcac aggcagactt caaatcaagg 11760

cagggaggga gcaggggaga acacccagcc tctctctccc tctgcccttc catcttctgc 11820

caatgcctcc cactggccca acccaacaga ggccagggaa gccgaggggt gctgtcccat 11880

ggctcaggct tctagagttc agaatggagc agaggagtgg tgatctgaga aacagcaaaa 11940

gtcaaaacaa tcaccagacc cctgagctgg gttgtgttag gagctaggga tgccactgag 12000

accttagagg tgtgtgagtg tgtgcattgg ggagaagggg taacccccct ccatggagct 12060

gaaggtcggc tcagcagccc aggcagtggt actgagggag gccagagcca ccccttctgc 12120

caggagtgtg gcggcattag gaaggcttcc agggacaggt gacactgggg ccagacgtga 12180

aagaacaggc agatgcagaa gggaaaagga agctcctcca aggaatgagg gttccaggca 12240

gcaccctgga catggggcca ggccacgggc ctcggaacta cgcagtgtcc tggagaagcc 12300

agccaagccc ttgaattctg acccctctaa ttgagaactt gggtctcagg attgtgcaga 12360

gagagcagga gcagatgagt caaagacaga agatattcaa aggaaatggc tggtacaaac 12420

gagactggga ggagaaggct gaagtggctt cagagagcct gttggccgcc cgagggccct 12480

tcagcagcct cgtttaccca caaacagctg atctgcttgt tacaaatcat tccagcctca 12540

ttagctacac ctcctcctga tccctacctg tttgttagct tagttctaat tatatgagag 12600

tcataaaatg ctttaaaaat aacctataac tcacagttct tggaaactct ggcgggtgcc 12660

tcccctgagt aatgtggagc ctgggggttt ttacgacagc ctggggttgc caaggggaaa 12720

ctgaggccca agcttgtccc cagaggagtt ctggctgaag tggtgtcaca agctccatct 12780

ccagcctcag tgaaggagtg ccccttcaag aaaccggcat gggggaggat gtttcaaagg 12840

tctactatgt accgggcact tcacatgctt tatcttctgg gaagtcatta tgcttcccat 12900

tttacagata aggaaactga ggcctgagga tgtgaaggga catgctcaac atcccacagc 12960

tggttgaagg cagaaccagg atttgaactt gagtctgtgt tgttcagaac ctgagctcct 13020

tctcctaagc cctgatgctc tacagtaccc ttcaccctgg ccttgagctg atgccttcat 13080

ttcgagcctg gcctttgacc ctcacctcac tcccttgggg aggccctgtg cctggcctct 13140

ctgagtctca gcttctgcat ctgtaaaatg ggggcccagt agtccttgct ggccacctcg 13200

ttgccagggt gcaggagcta atggattcat gagcagctct ccacagccct gagtgccagc 13260

atccacgtgg aagggcggct gttagcttga ttacaggcgc cgcagcagct tccgggaggg 13320

gaagtgttta attgggggtt ttatttccag cagcagggtc tggcttaatg aaatatccct 13380

gggcctcctg gcatgtggct gccctgctgc ctccctgtgt tgacaagcct tgagccaaat 13440

ccctggctgt gatttaccag ctcctcccaa gtcagataaa gctgggttga taagtagggc 13500

tatgccctga cccaccccac ctccagccaa ggtgggcatc tggggatcat gggcccagcc 13560

aggaaccagc ctggctttta atatttgatg ttaatgaagg gtaattgaca gatgcacttg 13620

cacaggctca ctgagcgagg gacacagtga tcctttgact ctgtgctagt aatgcacaca 13680

tctgtgcaca caccacctcc ccaccctccc aactggctct gaacagaggg agcaggaatg 13740

gtgcagcggc cgggggtcca cactggcaag ggggcctgct gggaggagac tgaatgttga 13800

gaaatctgag cccagaggaa gggcagagga gaatggcaca tgccacattc tttcctatta 13860

acagcaagtt tggctgctaa cttgggacct ccggaggcaa agagctggcg gattttagtc 13920

ttgactctgc tactttctag ttgatgatct taggcaagtt gtttggcttc tcagaggctc 13980

agtttcttca tatgtacaat gggactaact gttcctacct tgaaaggtgg ttctaatgtc 14040

caaaggtgtc aggtgtacag taggtgttca aaaactgatt ctgtgaaggc acagcctatg 14100

gaaagagcaa gtaaggagca tgttgtgaag gaatctgcaa caaggcagtg cctactgtat 14160

gccaggccct gtgccagctg tgggaagtgc atcttactag catcacacgg ttgcaggctg 14220

agtcctgggt atgcgggtct ttagatggtt tgtgtgcctg tggttccatg gctgtgtctt 14280

cttttctctg gtccctcaac cttcttcctt tctccccatt tcctcccatc aaagcaggct 14340

gctagtgcag gctctgacac catctcattc cgcatggagt cagctcaggt ccccatcact 14400

gcttttgggg gtatctacag gaaaacactg attttgcctc acgtcagaag acttggcccc 14460

agtgaagaat ctcaggcatc catgcagcag ataagagagg catttgtggg cttgagaggg 14520

gcccttcgcg ttgctaaaat cagaccacgt gcctcccctt ctctgccgtt gggggtacac 14580

ctgattttgt tcataaatgc gttcgaaggg tattggaaca ccatcgttcc cactgctgtc 14640

agtttcacca ttagattttc aggtttttgt ttttctccca ggtgaagtat cttttaaaat 14700

gcaggttctt ccagaaggag tcatgctgca agctgacttg ccccactgtg ttaccagata 14760

atttctgtgt cttgaagggg gacctggttc agggtttgtt tgggaaagga gctgggaagg 14820

aggagaagaa agagcaggga ggctgtcctg gtgtctggga tcagggttag gggagggcaa 14880

ggtccattgg caggccttgc tgacagtgct ggaagaggct gtcaccagac tctgcagtaa 14940

agttgtccct gccacatttt ttgctagggt gaaaaataga actcccagac tagatggttg 15000

cccctgtcct gcacccctga gggtcaggcc ctggggctgc cacagtcccc atcgtgtaca 15060

cacacacaca cacacacaga cacataacac acacacacac acacacacac acacacacac 15120

accccacatt aatgaagcct cttctcagga gactcagttc tttttttttt tttttttttg 15180

aggcagagtc tcactctgtc gcctaggctg gagtagttag tggcttgatc ttaactcact 15240

acaacctcct cctcccaagt tcaagtgatt ttcgtgcctc agcttcctga gtagctggga 15300

ctacaggcgc acgccaccat gcctggctaa ttttttgtat ttttagtaga gatggggttt 15360

caccatgttg cccaggctgg tctcgaactc ttgagctcag gcaatccacc tgcctcggcc 15420

tcccagagtg ctaggattac aggcatgagc caccacaccc agccgagacc cagttctgac 15480

tgctgtctct ccacatgaac actgagcagc cactgccccc agtgggcctc tggtgccttc 15540

tccattctct ggccttcact ccctccttcc tcccacagct gggaaaactg gtctttaaaa 15600

tcttctagtc ttttagaatg ttgaactctt ttaaagtttg tcctggctgc agcttctctc 15660

cttggtcccc ctgttcttcc tacaggtcaa tatcccccca ccctctcatt gtgccctttt 15720

gtcccaagtt cattggccct ggtgtcaaac ttaagttccc ctacagagag gccaggaatg 15780

aaccatttcc tcctggacac aagtaagcag tggcctctat cagcctcaca tcaaacctga 15840

ggccacaggc cagccatctc cctggtaggt ggacatgggg catggtgggg gacagcctct 15900

gctgggatgt gaatgggcac aggcaatgcc ccgccttgcc tctgcagggc ctgtgtttga 15960

tctctgttag tttccatcta tgctcttcct ttctcaggtc ttttttgttt tttctgtgtg 16020

agtcttgatt ctctgaagac agggacggtg ccaccatcct cagacagggg agctccctga 16080

ggacgggggc tggatccctt tcctctctcc aagggctatc tccatttcca acctataatt 16140

agagtctatg tgaaggtagg agctgggtat tccctttttc tatgggggat ctccaaaggc 16200

caaggctgca tacgaccaag aggccgtgta ctccttttag accagggggt ccttgcaggt 16260

aggtctgtgt ctcctccatc agattagaac ttcttatggg cagggctgtg tctctctctc 16320

tctcagatca gtgctctgtc ttccttccct tctcttggga actcagtgcc aaccttgttc 16380

ccagggtctg ctctccccaa ggagtgtggg agggaggtgg gcactgagtc ctaaaaatca 16440

gaaatagcga gtgactgcgg ggcaggcacg gctgctgctg gtcggtgcca gaccttgtca 16500

gtagtgtgat ttgaggctgg aggcagggac atggaactga gctaaactcc tgtcatctca 16560

aaggcatgaa ttcattaaac tgtgtcctag ggttgccgag aggcagggct gagggtggag 16620

ctgaggagcc cctgccttgg tggccccctg tcttgtccct ggaccaacag cgtatatgtc 16680

aggggaggaa ggtccaggtg tgtgtgtgtg tgtttgtgtg taaagaaggg aaaaggatgt 16740

cactaactca gagtagtcca ttctggggga gcagggatag ccctcagaat ggggaggaag 16800

gggagcatct agcactgggc aggctgccct attctgggct gaggctcagg gccaggtctc 16860

cccaccccag tgccgcaggg ccacgtgtac cggttctgca cagctgaagg cctctggctg 16920

cagaaggaca actccagcct gccctggagg gacttgtcgg agtgcgagga gtccaagcga 16980

ggggaaagag tgagttgagg cggggttctg agccagggag cggggagcca tgtcttggag 17040

cacttcactg gagcaaagac ccttggcttt gatgggggca tctgtggtca tttcatccat 17100

ctccttgcct ctgggggctt tgcacaccat gctttctgga caaaggtggt gtgtattcac 17160

ctctctggcc ttggaacagg gcccaagata tccaagaacc atcgccgtag gttacggtta 17220

ttctctttct tggtcttggt atccccggtg agtcctgact tggggcccca gctctgtctc 17280

tgtgccatgg gaaggaagat tgtgggaaga gggccatggg ctgcccatgt acacgtatgt 17340

cccccgtgtg ccacagagct ccccggagga gcagctcctg ttcctctaca tcatctacac 17400

ggtgggctac gcactctcct tctctgctct ggttatcgcc tctgcgatcc tcctcggctt 17460

caggtaaggt ggcccggacc ctgggagggg gctgcttcat cctaactccc ccagatagag 17520

gaatgaagca gcccaacacc aaatcaaagc aacagtgcag cacattcgtc cttctctcaa 17580

gagctctcta cagagccctg accgtagaat atgaggttga aaacacagga tgtgatactg 17640

tgttcttggt ctagtctcca cggtgtaaaa gtgtgcgtat gtactgcaaa aaagaacaaa 17700

aataccacgc aagcaccacc aacctcaaaa ccttaaccgt cggtgatggg attacggaaa 17760

atcccaggag atcttctcca atgaacatgt gtaactttaa taatcaagaa ttcagtatga 17820

ttttagaaaa gatcttgggg attagagatc ttcctccctt tggagctttt cagccccagt 17880

ggaaaagttc ctccttagat ctaacctaga tgagacgttc cttccaaact aggagctggg 17940

aacagagtac caggttcttc ccactcctct gcacttggag aaggggggcc agggggactg 18000

ggttcaaaga gggaagggat tctgagagaa tgagacctca ggacaccagg cagcaagagc 18060

aggagaggtg gcattccctc tcctaccttc aaagcacctg gcacagtcct gggcacacgg 18120

tgctcaataa atgcattcac tcaatgaata ttctcctctt ttattatgaa ttattccagt 18180

gattcaaaaa tgcaatgact gatactatga tcactcatgc acacaccatc cagcctaaga 18240

aataacatgt cccatagtta cacttctctg ggtaaccctc tctgatccca tccctctccc 18300

tgccctacac ccatcagagg taagccctga atgtgcctct tttaattccg acacatttcc 18360

ttgtactttt accacatttg tgagtagcta tagaaatata tagcaaggtt ctgtgtattt 18420

ttaatagtgt attgaacata tttgtcttag tcttttcctg ctgctatagc aaaatacctt 18480

agactgggta atttctaaat aacagaaata tattcctcac aattctgaag gctgtgaagt 18540

ccaagatcaa ggcaccagca gattccatgt ctagtaagtt cttgtcctct ctttccaaga 18600

tggcaccttc tcactgtgtc ctcacatggc agaggggcaa atgggtgaaa agctctcaca 18660

aacaccctca ggtctcttat aaggtactaa tcccattcgt gagggcagag ccttcatcaa 18720

ccttatcacc taaaggcttc accttttcat actattgcct tggggactaa gtttcaacat 18780

gaattttgga gtgacacaac attcaaacca tagcattctg cccctggccc ccccaaattc 18840

ctgtcctcct cacatacaaa atacattcat ttaatcccaa tggctccaaa gtcttaattc 18900

atcccagcat taactttaaa gtctaagtcc aaagtctcat ctaaatatta tttaaatcag 18960

gtatgtgtga gactcaaggg atgattcctc ctgaaggaaa tttctctcta gctgtgaact 19020

tatgaaatca ctgaagttat gtgcttccaa aatgaaatgt tgtggcaggc attctcattt 19080

cataggacag acattcccat ttcaaaaggg agcaataggt ctgcatgtgg tggctcatgc 19140

ctataatcct agctaattga gaggcgaaag tgggaagcct gcttgaggcc agagattgga 19200

gaccagcctg accgatgtaa gagaccatta aataaataca attgtttatt tttttatctg 19260

gaaaaaaata aagaattaaa attcaaataa aaaaagagag aaataggaag gaagaaaggg 19320

gtaactggtc ccaagtaaat ccagacttaa tgggacaaac aacattaaat cttaaagctt 19380

gagaataatc ttctttgact cagtgtcctg ccttcctgat atggaggtgg gagttgggtc 19440

cccaagtctc cccgaagccc cactcgcaca gctttgctgg gttcagctca tgtggcagct 19500

ctcacaggct ggagttgtgt gctggtggct ctaccagtct ggggtcttgg aggagaatct 19560

tgccccccat gactctgcta gtcattgccc tagtcaggac tctctgtagc ggccccacac 19620

ctgtgcctta actactatag atttattaga aacctccact ctgcctgggc cctgaggctc 19680

tccaggatat cctttgaaat ctaggtcttc ctggatttca aagaagtaac tatgttccct 19740

acagctcgtg cactctgtgt gtctgtagag ttagcaccac gtgaacactg ccgaggttta 19800

ctgctacgtc ctccagaggg acggcctgag ttatacctgg gcccacttga gccgcagcta 19860

gggtagccaa ggagcactgc acccaagtgt agggagcaga gacaggaggt ggccctgggc 19920

agtgacttcc aatgcctcac agtgtcctgg gctcctcccc cagaactgtt ctgctctcaa 19980

gaccctgaca tctggacctg aaagatctct gaagtgcatt cagggtcatt cttccatttt 20040

ctacccatac taattttatt aaatgttcac ttggccacac ccttggtatt gtctcctgaa 20100

catgcttttg cattctttac aatatggtca gactgaaaat tttccaaatc tttaagttct 20160

ccttctcttt tgattataaa tttcatcttt catttctctc ttctcatatt ttgctataag 20220

cagtcaaggg cagccatgct gtaccctcaa tactttgctt agagatttct tccaccaaat 20280

attctatttc attgctcata agtctttgct cccatgaaac acttggacat agacacaatt 20340

cagccaagtt atttgccact ttgtaacaaa aagatggcat ttcctctggt ctccaatgcc 20400

atgctcctca tttctatctc agacctcatc agaatggcct ttactgttca tatttctacc 20460

attattatgt tcacaaccac ttagataatc tctgtgaaga ttgaaatttt tggccgggcg 20520

cagtggctca cgcctgtaat cccagcactt tgggaggccg aggcaggtgg atcatgcggt 20580

caggagatcg agaccatcct ggctagcatg gtgaaacccc gtctctacta aaaatacaaa 20640

aaattagctg ggtgcggtgg cgggcgcctg tagtcccagc tactcgggag gctgaggcag 20700

gagaatggcg tgaacctggg aggcggagtg agcagagcag tgagcagaga tcgtgccact 20760

gcactccagc ctgggcgaca gagtgagact ctgtctcaaa aaaaaaaaaa aagactgaaa 20820

ttttctacat agctctcatc ttcttctgag ctctcaccag aatcaccttg tatggcccat 20880

ttgtggaaat atagactttt ttctatccca ccacccagtt cccaagctac tgctacattt 20940

ttaggtattt gttacatgac tccccactct cggtaccaat ttctatctta gtctgtgcct 21000

gctgttataa caaaatacct tagactgggt aatttttgaa aaatagaaat ttatttctca 21060

taatttagag ggtgggaagc ccaagatcaa ggcaccagtg ggtttgatgt ctagtaaggg 21120

cttgctctct gcttctgaga tggtgtcttc tcactgtgtc ctcacatgat ggggaaggag 21180

tgaacactat gccctcacat cacagaaggc aaaagggcaa aaagggctca caaactcact 21240

taggcctctt ttataaggac gctaatccca ttcttgagag cagagccctc atgatctaat 21300

catcttctgg aggcttcacc tcttaatact atcattgcat taaggattaa gttttatcat 21360

gaattttgga ggaacacaac attcaaatca tagaaatatt tttctgtaat tttttggtcc 21420

aacattatgt ttgtgtgaga ttcattcaca ttgacacgag cagtcctgtt ttgtggattt 21480

ttaaccacca ttccatattc cgttgtataa tacagcacag tttatctgtc aaccttttaa 21540

tggacatttg agttgttctc tttttttctt tctgaattac aaatgttggg tgcaatgtac 21600

attcttactt gtgcatttct ctggggcata taactaggag tagaattgcc aggtctgact 21660

accacctgct ttcgcaattt tactatgtta agttgttctt cacagtggtc acaccagttt 21720

accctctcat cagtaataaa agagagcttc ctttgtctag ccttctcacc aatatttagt 21780

gtcttcagac ttaattgttg ctaacctgaa gtatgtgaaa ttgccttatg attttaattt 21840

gcatattcca gattcccaaa gagatcaagc attttttttc atgatttttt ctgtgagttg 21900

ctttttcata tcctttgctt actttttcat tggcttattt gggtttttct tttctatttt 21960

acatattcat tatatattct ggatacttat ccaggattta ccctctcttt agtctgtggc 22020

ttattttttc actcatttta tggtttttta tgcacagaag ttcttcattt taatggaata 22080

aagtttatca atttagtatt gataaataaa atttatcaat aaagattaag cagttgataa 22140

cagttaattt tattgattgc tttaaaatta ttcatttttt ccaaagaaca aattctggac 22200

agtcttatta agaaatcttt ccttggctgg gtgcggtggc tcacgccagt aatcccagca 22260

ctttgggagg ctgaggtggg tggatcacaa ggtcaggaga tcgagaccat cctggctaac 22320

acagtggaac cccatctcta ctaaaaatac aaaaaattag gtgagcatgg tggcacccgc 22380

ctgtaatccc agctactcag gaggctgagg cgggagaatc acttgaaccc aggaggtgga 22440

ggctgcagtg agccgagatc acgtcactgc actccagctt gggcgacaga gcaagattcc 22500

atctcaaaaa aaaaaaaaaa aaaaaaagaa agaaagaaag aaaaagaaat ctttccttac 22560

cctagtgtca taaagatggc cgtctatatt ttctgtaagt ttcagagttt tgctcttcct 22620

tgtggttctt tgatctgctt ggaattcata ttgcgtatga tataaataaa ggatttcatt 22680

tttttttctg tatgtggaat tagctgcctg agtaccattt attgatagcc tttccttccc 22740

ccttggtttg caatgactgc cattccatgc agcaaggctc cacgtatgta tgggttggtt 22800

tcagtcacct acagtttttt tctgtttatc tttccctgtc cggataccat ggtgccttag 22860

ctactgtaga tttactagaa atcatatagc aaaatctcct cctctgtgat tctttaggag 22920

tgtctggaat attcttagcc ctggctcttc tttataagtt tttaaagttc tactttaaaa 22980

ataaggtaga atttttatga atacggagag acttgactcc tttaaggtag cgagtctttc 23040

tatctgtgaa tgtgatgtat ctatctccca ctagtttaag tcttcgctaa tgattttaag 23100

caagatttat aaaattctcc atcgagatct tacctatctt ctgttagact catttcaagc 23160

tagttaatta aaaatattgc aaaagaatta tttctaaaca tttactttta aattttttta 23220

acgcaggcat atacaaacat acttatcagc aaccttgcta aactgtctta ttagttctaa 23280

taatttgtcc atagatttct tgaattttct atgaagataa tctaccatct acaaataata 23340

actattttgt ttacttctga ttcttgtaca tttttccctt gccttattat agtgggcagg 23400

atctgtagtt acaatgttga ctagaagcag tgataacaga catccttatt ttgttcctga 23460

tttcaaagga aatgctccca cagtttctca gttaagattg atatttgccc ctggttaggt 23520

aaatagcctt cattaggtta agaaatgccc ctgtactttc aatttgctac acattttcac 23580

atgaaaggat attaaaactc tatccaatag tttctgaatt cctaccaagt gcctagtcct 23640

gctctgcctc tgtgaggcat ttaggatgtg ggccctgtca acaagaagca gtgttcccac 23700

tggggaaact gatcctcatg cataaatctc tttggttatg caagactttg tgtcaaagaa 23760

gatgctgcag agagaaggat agaatgtgga tgggaggagg gggcagtgac tacatagcgg 23820

aagccttcat ggagaggcag gatttgagtt ggacagaagg attttcacga tgatgaggaa 23880

gaagggtatt catagaagaa gacacacgca cagtcccagg gctggaaaca tgcctaatgt 23940

gtgtgttagc ttgagagcct ggggttgtag agcagaactg ttggctgcct tgccagggaa 24000

aggccctgct ttctccctca gacacctgca ctgcaccagg aactacatcc acctgaacct 24060

gtttgcatcc ttcatcctgc gagcattgtc cgtcttcatc aaggacgcag ccctgaagtg 24120

gatgtatagc acagccgccc agcagcacca gtgggatggg ctcctctcct accaggtgtg 24180

tggtgcatcc aggaccgcct caggagggat gggcggttgg aggaggcctg cctctgccac 24240

cctagacagg cctgggacag gagaagacaa gagccgaaca gtcctggccc ttcgggagcc 24300

cccagtctga tgggagaggc agttcgcctt gggaccctcc agtctgctga gagaggcagt 24360

ctaccttagg gagctcccaa tctaagggag gagacactgc cctagggaat tctcagtctg 24420

acacaaaaga gtagtctttg gagacacact tgagtttgaa tgtcagctct gtcacttact 24480

atctgtacac ttttaggcaa gtcacttact tctctgaggc tgagaaatag gtgagaagtc 24540

acttacttct cacctgcagg gggatacatt actttcttcc aatcaggata gtgaggattc 24600

aatgagctaa agcagataaa gtccttagca ctagccctag ccagagatgt gagctctctc 24660

ctcctgcatt aaccatggca ggatagtggc tggagcagga cggggagtgg tatcaagaga 24720

ggcagagggc agagctgttg tccccatgac acccttcctc tacccccagg actctctgag 24780

ctgccgcctg gtgtttctgc tcatgcagta ctgtgtggcg gccaattact actggctctt 24840

ggtggagggc gtgtacctgt acacactgct ggccttctcg gtcttatctg agcaatggat 24900

cttcaggctc tacgtgagca taggctgggg taagaaccgc catcacccac cctggacctg 24960

tggcacgggg gtgggagacc ttgacccctc ttctaacatg gtacttggaa cgcactgctg 25020

agcaaggggc aaggcccact gggtaaccct cttcctgggc acaggacctt ctgccccggt 25080

gctgttaaga tgccgtaggg ggttatggtc ctcccctcct ctgcataact ctcctccctt 25140

aatggaagcc tccaccgcat gtggccaaac accaccctct accactatca tgcacaccac 25200

tggaggtcat ggcactgttc tcttaagacc acatggggac ataagttcac acccagtccc 25260

gggcagcccg agggcctgag ggaaatggtg agtcctgggc ctcaaggtgg ggaggggatc 25320

aagagggtag ggggctggct ggggggagcc tgctggtgct ctgtcacatg acgatgaaga 25380

ggagggctca gaggagagcc aagtacagtg gggcaggggg gccagaggcc agccaaggaa 25440

agggtggacg gcggatgtgc ctggagccca gctggaaatg ttctagccaa aaaagtttgc 25500

caagaaccat tgtgtctttt tttttgctgg aacatttctg gttgtgcttc tgcctctcac 25560

cacgtgggat tgtaatgagg ggcagcattg gggttgggaa gggggctggg atccccttca 25620

cccctgggaa aacatgcaac aggcctttct ggagggaccc acggtaccca gagaataggc 25680

cctgccctct gtcctcccta atctgccatt gaatccatga gttttcctgt tccacaccat 25740

cccccgccct gcttgccact ccttcattca ctcctccttc cttcatgcgc ccacctcacg 25800

ctgctctgac cacggctgcc cctgccacgg acctccgggg actttccagc tcagtcacat 25860

ccagactcct cttccctgac aatggtcctg tccctgagcc tttgcaaggc tgttctccat 25920

gcaggaatgt tcagggagca tccttcccac accagggtgt ccgctgcctc ttcaggaagc 25980

tgcctcaggc caccagtcct cgtggttctg tgctctctgc cctctcaagc tctccatccc 26040

ctgtccttgc acttcattta tttttactgg tgttcctcag ccatttttct ctatacagct 26100

ccccccaccc cccaggagaa gagtagcctg agagatttta atgtgaccag tgtcttgagg 26160

acaccaaggc ccagagctgg cagggcctta caggcagagc aggccctgga acccaggcct 26220

cctgactccc aggctctggg cctgcggcca tcatgccctg ttctcttgcc tgggctgcat 26280

cccttcagtc ccctgcagca cccctgagca ggctcaggcc tctgcacagg tgggcaggta 26340

ggcctggctc ctgctcacag atccctgctc ctcacctgcc tcctccactc ccagaaccca 26400

gtcccttcct tggccttttc tccccgaggc ttcttcctcc tgctagagca ggaggaggga 26460

ggccatcgag ggcagctcag ggaaggctgt cttcagggtg ggggacggtg tctgtccatt 26520

ctaggaaata gcatcagcgc tgttagtgcc tgtcaccatg gcgacccgag ggcaggaaca 26580

atcaagggac gtgtgtgatg tgtgcagagg agcgggggtg gctggagttt ggtggggaca 26640

cactgcccta gaggtctctg agtgggagat gggggaggga gtgccaagtc cttttcctac 26700

cacctcacac cctagctcag ggctcggggt ctgtgggagc acaagggcaa gacccggagg 26760

acaccctgct ctaatcagaa cacctttttc ttgaggattg gggtggggga gacggagaga 26820

gattcacagg ttgctaggag ctcagacgtg atgccaggag gccttgccca agctgggata 26880

cttgggaaac aactttcagc ttcatgtgca agtggaagaa ttaaaattag gactctgaca 26940

cagttggaga ggtcccccaa agggcatggc agcttctgct gcagtcccca ggcagttggg 27000

ggcctgtaga aacccacatg gaggctgtgc aagagggagg caaatgatgg ttgagggaat 27060

cattacttcc tggagccagt tgtgtgtgtc ttaatttgag cttttttctc caacctgtct 27120

tgctcgtttt ctaaatggga gaaagatgaa gccagcaggg agtgcgtaat gtttgcatgc 27180

agcagggctt tcgtgactgt gttttttgaa aaggccttgt taaggaaatg ctgacgatga 27240

gtgaaacttg ccaggtctgg ctgataacat gggtgagtgc cctgatcctg gaaggaggtc 27300

tcagagagaa gggacagcag ggggcgaggg atacccagaa ggaagcttct gcatgtgtac 27360

gtggttcagg aaatcggcat gctcttgccc agacaatgtt ccttccctta ttggtgtgtt 27420

gactcaccca tcattcgttc cattctcact gggcatcttc tatgtggcag ggcctgtacc 27480

atgtgctggg gcctttggag accatcccat tccttgtcca gtctcataaa ggcctgttac 27540

taaagcaccg tagatgctga gaggtccgat ttgtgtagaa atcaatgggc caggagagag 27600

agagctgatg aagaatgctg gtccgctggg ggctcctttg cctcctccat ctctccctgc 27660

atcatcaggc acagtagatc tgggcacaca acctgcttag tagctgctgt tgagtggaca 27720

ctgatgtgct aaagacatag acaatggtga caggaaaggc tatgaagtgg ttctgtcctg 27780

gaaatgtgct ccaggcagag attcccaaag tcatacgtgc acactcatgg tccacccacc 27840

acaccctcac acacacatgt ctacatacct taatatgcac atgcttacct atctcagaca 27900

catctccaac actgagggag gggccaacgg agacgagcat ggagacagcc agtgcagaag 27960

gccattgagg gcggtgggag tgggtatggg attgcagccc tgaattcgag ccccacctgt 28020

gcccttctga gtccctgatc catctcctca tatgtcatgt gaggaaggaa aatccaattt 28080

taaatcaacc aacacagacc ctgagctctc agggtgatct gggcattgag gctctgaagg 28140

tgagtgaggc ccaggctcta ccctcaagga actgggaacc agttgtatta attctctatt 28200

gctgcagaac gattaaccct gaaacttagc tacttaaaac aacgaaccat tgttatctca 28260

cagttcctgt ggcctgcaat ctcggttcaa cttagctggg taccttgtgg ctcaggatct 28320

cacagagaaa actctttagg caagagcatg tggatttcct gaatcacata cacatataga 28380

aacttccttc ttgggcatcc cccaccccct gctgtccctt ctctctgaga cctccttcta 28440

ggatcagggc actctcagag aagcagccag ctgtcaccag ggttgctgtc atctcaaggc 28500

ttaactgggg atggagaatc tacttccaag ctcattcaag aggcagttgg caggaagccg 28560

cagttgctgg ccaaaggctg gaggcctcca tttcttgctg cagagacctc catctaggcc 28620

ttctgagttc tctcagcaca tggcaactgg tttcccccag agcaagtgat cagagaaaga 28680

gaaagagaat gcaaaacaag ccacagtgtc ttttataaca atcagaagtg acatgccatc 28740

acttctgcca gaggttcctg ggtcacatga aacaatgctg atacaacgtg gactaactac 28800

ccaagggtat gagcacagga ggtggggatc cctaggggcc atcttagagg ctgtctacta 28860

cacaagtcaa gtctcagccg agtggaaagt agtattgttg tagctgttgt tattgtaggc 28920

cactgcggac agttagttgg tcagctggca tcaaggactg ggctcctgtc tccattccag 28980

gattctgccg agccctcttg aaggtgcaca gtggtaggga agagatgact gaaaggacat 29040

ccagtgcctt taggagttga ctggcaacgt gggctgaagc aagagagaaa atgggcatca 29100

gagggttcgc ctgctccttg cctcatccgt catgctgtcc ccaccgcatg gcccagtccc 29160

gctgctgttt gtgtgtctgt ggcctggtaa cttagccggg actctgaggg caagtcctga 29220

cagtgtgcct ggcacacagg aggtggggga ctgatgactg atgaggacct ctctgtaagg 29280

aagagtctgg gggtccgcat ggggctcctg ctgcctctgt tctcccaggt gcccagaaaa 29340

aaactagaaa acatagttgg aggccaggca aggagagggg ctggaagtgt gaggggcttg 29400

gggcagggag aggcctcggc atgtagactg tggctctggc ctgccagccc ctctcccctt 29460

ctgtctttcc ctctctctta ggtgttcccc tgctgtttgt tgtcccctgg ggcattgtca 29520

agtacctcta tgaggacgag gggtgagtgt cctgctccaa gggggcgggt gtgcccaggt 29580

ccccatcctc aaggtccatg ggattccttg gtacctgggg cacccatctg ttgagaggat 29640

aaaggggggt accaggagct tagaaggtga atttagttct ctaatctccc ctccctgccc 29700

ctgcccctgc ccctgcccct gtcccaggat tgtttgatga ctaacggaac tgggtctgtg 29760

gggtacatgg tgccctcctg caggtttttc tgtaatatct gcaagtgaaa tgtttgtgtc 29820

attttgtata tgatgctggg tacacacatg actgtgttgg taggtccttg aaagcttgtt 29880

tgggggctgt gtgcatctct gtgtatgact gtgtatttgg actatgcgtg tgtgttgtgt 29940

ggctgtgcac tggcagggct ctgtgggtcc atgggactgg tttttgggtg gctcctggga 30000

gctgtgtgtg tggcactcaa catggccatg tgcattggcg gcctctgtgc ctgcacctga 30060

gctgggggtc ctgtgagtcc tgctggatgc atgtgtgcat ctctctccct ccccagctgc 30120

tggaccagga actccaacat gaactactgg ctcattatcc ggctgcccat tctctttgcc 30180

attggggtga gtgatggtgt cagggatggg agtggcagct atgatagggc tgggctggtg 30240

ccccctgcca atccccggcc ccaccccgca ggtgaacttc ctcatctttg ttcgggtcat 30300

ctgcatcgtg gtatccaaac tgaaggccaa tctcatgtgc aagacagaca tcaaatgcag 30360

gtgatgtaac tgagctggct ttactgagga ccctcagcaa gtgccccttt ccttctagca 30420

gagagagaga gagagatcct gggatgctta gcttagagcc ctacactacc ctctccttcc 30480

acccaggcct ggccttggac cccaaaccct tgcttttgcc ctgtctctgg gcagcccctg 30540

gacttgttgg ggtcttgacc tctattcttt ccctccaggc agcctgtccc agggtatttg 30600

gggtgggaga acatccatga cccagatatc aggacttggt aggaagtggg gagggtagag 30660

aaagggaaga agagtccatg ggagaggtcc agaatgactc gagatctctg ccctgcccct 30720

cagacttgcc aagtccacgc tgacactcat ccccctgctg gggactcatg aggtcatctt 30780

tgcctttgtg atggacgagc acgcccgggg gaccctgcgc ttcatcaagc tgtttacaga 30840

gctctccttc acctccttcc aggtgacttc atgcttgggg acacttgctt gtaaagtcct 30900

agagtggggg tgggacagac accagcctgc atcatgcaga tggaaaaggt gggaagactg 30960

ggacctggag gggtgatccc tgcccaaagt cacctagttg gagcagagcc agaactagta 31020

tccccaggtg tcccaatacc tgggccagtc ttctctgtaa gcacatggga caagaagcct 31080

tcctggatgt agctctgggt accagcctgg cattgcagct gccatctact tggtggcgag 31140

caccctgcca ggtgctttac atgcattgtg catttactcc tcacagaacc caggagatgt 31200

gcagggttag cctcacttta cagaggagac tcagaggtgt taaaaccaga gttacggtgt 31260

cacagtttca aagaaaagaa ctgatcctct ccactagcat tagatgatgc taagggggtg 31320

taaaaataca aaaaaactgg tccctgcctg cccctcaaga gtgtcatgag atgcctgcac 31380

acatgtgggc tcatgtgtgt gtatttacat acatgtgcag acatacagaa cacaattctg 31440

gagccctggg tctccttcat gaacagatgg ggctggagtg gggaatattc tccatgatca 31500

gagtcattgt acttcactgg atcaggcctc ctcaaggaga ggttggatgt aggatctcta 31560

cagcttctgg gcagcttctg agcagggtct cctggacccc atggcacatg cctgggagac 31620

ccatggggag aggctagctg gggcctgtag ccaggcctag tcgcagaggt gcaaccctgg 31680

tgttccttca gaaaggaaat gagagggaag ccactgcccc attcctggat ctgcctgggc 31740

cgctggattt ggggaggggg ttgttgagga tggagtccca aagtgcttcc gaccagggcc 31800

agtctgcagg gcacctccct cctgcttcct ccctcttgat gtgactcttg tttccagggg 31860

ctgatggtgg ccatattata ctgctttgtc aacaatgagg taagctctga ggagggacgg 31920

tggggaccct ggtgggtggg taccatcagc ccgtctggag tagtacaatg gggactccca 31980

tcaatcttgc cagctgaaac cccctactca cctcactgag gcactagggt tgagaagcca 32040

tcttgtccac agatttggtt ttctgtgcat ggtctctctc acacatgaac acacacacac 32100

aagtacatgt acaggtatac cctaatggca tgtgacccta aggaagaccc agactcatgt 32160

taaggggatc ctgaatgacc ctctttgctt ggtggtagtg actgtcactt acaaagtgat 32220

tcaacacaca catacccaag ccacatgctt ggggctttaa taatcactat ctcatttaat 32280

tatgaccccc acatccctca aaaaaaaacc ctgctaaaat tcttatttca tattcccatt 32340

ttttatatat acatgaaaac actgaagatc agaaagagtc aggaacctgc cctaggtttg 32400

gaaatagtgg cacacatcgc ttcttagcct tttggctaag gtgaagtgtg gaaatagtgg 32460

catggcagag ccaggattca aattaattct gcattaaagc agaaagtacc ccattcttca 32520

tacacacagg caagagccct ggctcactgt ggggaagggg gtggctttct ggtgctagga 32580

tgggagaagg gggttctcgc tggccccaca cactctcctg aaaatgtctg caggacagca 32640

ggcctccctc ccatccctga gatggtcagc tcctgggagg agggcatggc tgtgttcatg 32700

ggcacctgga aaaggcccct tccaccaagg tactcagctt tggaatccca gagctcaggg 32760

atgttggagt cagcacatta ataggcttgt ctttttctat gggtgtgggg gatgcagaaa 32820

ggacagtggg tggggacctg ggtgtttcaa ggagctatga gatggctcaa agaagcaatg 32880

tgatgtagca gaaaagatgc aaggatgcag gctttggcat cttatttggt ctgatctggg 32940

ccccatcact taatgagctg tgtgactttg cctaagttac ctgcccactc tgaaacttag 33000

tttgctcatt tacaaagtgg ggataatcct gtgaggatta agaaagtctg aaaaaggtgc 33060

gaaaggggaa cagagaacaa gtccttcctt caaggtggga tgctgaaggc ccatcctagc 33120

tcctcacagg tgctgcttgg catggagaaa cacacaccat tatctccaga tgctcagtaa 33180

gcaatcacta ttagatcagt attcagtgat tcccagagac aggggcctaa ggaatttcag 33240

aggatgccgt gcagagaaaa tgaggctggg tgaaagggaa gggaggggaa tgttcccttg 33300

tccctgaggc ctccccctgc ttctgctgaa ctggctacct gtgggaagtg tggccctcag 33360

atctctaaat gggagggaaa ccaaggtctc ttaagggttt actgcatata aggtgctcag 33420

agtgtctcat tacatccaat gctgtgcagc agatggtatc cccaattcac agctaagaac 33480

gttaagcctc agcaaaatga aatgagtagc ctaattcaat agacagaaga tggtagagct 33540

agtgggctgt ggcgggggca agatcccact taatgcctca gggaagcagg tgctggggag 33600

gtgaaggcag cgccaggcaa agagtggccc ataaatggga ttctggctgc tcctgggaca 33660

ggtcttagag gacccagcgg ctccgtttgg agggtagggc agggaacagg ctcgtgaccc 33720

acaggaggca gtcctggtcc tcctccactg ccatatcctc aaaatgagtt tctttaaaac 33780

tgatgcaaac gaggaagtgc aaaaacctaa gctctgtggg tggcctcccc tccaggaatg 33840

ctctcagcca gctgggtgat gcgctaccca gccctgagct ctgctgcttt ccattccagc 33900

ccagtggtga tgggggcaag acagggtggc atgtatcctg tgtggggatg aggagcagag 33960

gcctgtaggg agagactgag gctccctgct gccaccttgt catcttgacc cccccacgtc 34020

cccgtttctc acttctctat cctctgacac tgtcactgca gccctactag cagacacgtg 34080

tgcttggctt cccccccgcc attgtgaaca gtctcccctg acccctggct cagctgcctc 34140

tccagattcc atccttccaa gcctagtcat ctcatcacct ccctcctcat gcaccaccta 34200

tggctggccc acctttgtaa ggggatagta cttctttgcc cttggaaaaa aacttgatcc 34260

tgaatcacca gggttgcacc tctctgacca ggcctggcta cagaccccag ctagcatctc 34320

cccgtggatc ttccaggcta gtgccacggg gtccaggaga ccctgctcag agaacgggag 34380

aacctgtctc ccttcagagc accagccagt tcctttaaac ccaccctgag actcctcatg 34440

ccctggctgc tccaggcaca aggacagata catcattgtc actgggccac aaaagacaca 34500

tgtggctctg gggctaaggc ctctgtccac aagagctgtt ctgcagaccc agggttgcca 34560

tggcaatgcc aggtacttgt gacctgagga gaaaagatgg cttgaatatt ggttctgtgt 34620

ggctgccaag gcctgcacca gctgctggtg ggcgtgagtg gggacaggga gggggaggcc 34680

tgaaggtggt tttgctgtag cttgtgcctg agagccacag tgaggtgact cagagccctg 34740

agtgcttgtc ccagccctgc cactgccgag ctgtgtgacc tggggcaagt cattttgcct 34800

cccttgcctc tgtctcccca gccgtaaatg aaggtgaatg agcttcccaa ttcaccccga 34860

atcctgaggc tggcatggtg cctgctggag agggaggtca ggaggggtgt gctgcactgc 34920

agaggtcgag ggcagggatg aggataaaaa tccgatggtg gaggaggaag tgtggtggcc 34980

taagtcccag cccaggtgct cagaatctgt ggtgttgtct ttgtgggttc attttcatag 35040

ctgaggaaca gaaggaacct gatgtgagat gctcatgcat ttatgtgttc attcatttgg 35100

ttcatttaaa acacatgtac tgagcacctg ctgtatgctg ggcactgtgc taggttcagg 35160

gatgctgaga tgaagaagat gtggtcccag ccctcgaggt gctcccaggt gagaggtagg 35220

agatggacca gggacaagca ggactgcaag agccacgcag gcaggaggcc aggggttagg 35280

agccgggcat tggaatcagg ccagatctag gttcaaatcc tggctgagcg acctgaggaa 35340

ataaacactc tgcacctcag tttcctcatc tttaaatgtg gagctacttt gcagggttga 35400

tgtgaaagtc agctaagata attaatggaa aattctgtgc atacagtaag tgcccaataa 35460

atatggtgat tttcaaggac aatcgtgatt acaggggtca cagcagaggt tcaggaaaag 35520

tgctgaagga gatgggggca gaaggagaca ctgtgtgtca gcaccctgag aagtcccata 35580

ggtgtccctg aaacctctct ttggtctctg gtttcccgga ggtgtgcctc agtttaccat 35640

ctttcccccc ctctccagcg gaagggctat gaaacaagcc tggcctcctg gcagctgcac 35700

aagtgggggc caccactgga ctcagggccc ccatcccagc tgtcagcatg cgctcccttc 35760

acaaagactc agccgggcag ggagctggca tacaacaggg tggctgtagg tggtgtctgc 35820

tcagcctcct tccctccccc actcaagctt gggttcatga cgctgcatgg gacactggcg 35880

tcatcctggc aggcagcctc tgggccatct caactctact cagtgcagaa aatgttcttt 35940

taagggatgg catgcagcca taccaggcgt cttgctgggc atagctggat ggacgacaaa 36000

ggaggagagg agggttgttt gcacaggggc tggtggggag attggcccct tatccacagc 36060

cgctggcatc tggctgtctt ggtgacttgg cctaaaggga agggcccaga ccccagacac 36120

tctggtgtgg gaagggtggg gacttcaaag aagctctgga aagcaaatgt atgggaacat 36180

cacccagaag agcagtccag accccccagg actagcttgg caggttccag ctaggcctct 36240

ccagggacca gaatcactgg ccaccaccgg gagccacagc attgccacaa accctcaagg 36300

gatgggggct ccacagtgtt acttggattt gtctctttct gccccaacaa tctggccccc 36360

aagcatcagc cctcctgaga ggcttttcct gatggggagg ccagagccca gcacaacagg 36420

gatcctggct gactgagctc ccctgggctg cacccccatg gtcactgcag tgtctctcta 36480

gatggcatca gttgagaagc cctcactggc tgccccttcc tccctgctgt ttcccgtctc 36540

agccacattc cttccttccc tccctcctcc cctgtgaggt gctggggcct ggagagaagc 36600

cctcagggag tttagaaggg tctgctgtag acaccaggaa ggctgggggt tgccgtcccc 36660

cttctcaagc atccaagctg aaactggact gtgcgctgta aagacagagt ctctcttctc 36720

tctcccatct cctgtctccc tgtgtttcac cctccctctc attctcttcc gttaaataat 36780

actgtgatgg attgtaaaag gtacttagct gaatcacaga gtttagagat ggaaaagacc 36840

aattaggtca tcaagctgat ctctctcctg aggagctgca gaaaatagga gacaacaata 36900

aacaatttta ttgggatatt aatttaggag cccgaaggcc ttgacttgtg tctcttaata 36960

taatgcagtg gatttcttcc cttccccact aatgtaatag tggggccatt ttgttagcca 37020

ttggtttgca tgatggcttt cgtttccctc cttttcccat ggaaggtcca gctggaattt 37080

cggaagagct gggagcgctg gcggcttgag cacttgcaca tccagaggga cagcagcatg 37140

aagcccctca agtgtcccac cagcagcctg agcagtggag ccacggcggg cagcagcatg 37200

tacacagcca cttgccaggc ctcctgcagc tgagactcca gcgcctgccc tccctggggt 37260

ccttgctgca ggccgggtgg ccaatccagg tgggagagac actcccaggg acaagggaag 37320

gaagggacac acacacacac acacacacac acacacacac acacatacat cctgctttcc 37380

ctccccaaac ccatcagaca ggtaaatggg cagtgcctcc tgggaccatg gacacatttt 37440

ctcctaggag aagcagcctc ctaatttgat cacagtggcg agaggagagg aaaaacgatc 37500

gctgtgaaaa tgaggaggat tgcttcttgt gaaaccacag gcccttgggg ttcccccaga 37560

cagagccgca aatcaacccc agactcaaac tcaaggtcaa cggcttatta gtgaaactgg 37620

ggcttgcaag aggaggtggt tctgaaagtg gctcttctaa cctcagccaa acacagagcg 37680

ggagtgacgg gagcctcctc tgcttgcatc acttggggtc accaccctcc cctgtcttct 37740

ctcaaaggga agctgtttgt gtgtctgggt tgcttatttc cctcatcttg ccccctcatc 37800

tcactgccca gtttcttttt gaggggcttt gtttgggcca ctgccagcag ctgtttctgg 37860

aaatggctgt aggtggtgtt gagaaagaat gagcattgag acggtgctcg cttctcctcc 37920

aggtatttga gttgttttgg tgcctgcctc tgccatgccc agagaatcag ggcaggcttg 37980

ccaccgggga acccagccct ggggtatgag ctgccaagtc tattttaaag acgctcaaga 38040

atcctctggg gttcatctag ggacacgtta ggaatgtcca gactgtgggt gtagattacc 38100

tgccacttcc aggagcccag agggccaaga gagacattgc ctccacctct ccttggaaat 38160

actttatctg tgaccacacg ctgtctcttg agaatttgga tacactctct agctttaggg 38220

gaccatgaag agactctctt agggaaacca atagtcccca tcagcaccat ggaggcaggc 38280

tccccctgcc tttgaaattc ccccacttgg gagcttgtat atacttcact cacttttctt 38340

tattgctgtg aatagtctgt gtgcacaatg ggcaattctg acttctccca tctagtggaa 38400

atgagcgaaa tcatggttgt agtgatgttg tttgggagag tgcagtagta attgatttga 38460

cccactcaca cttggagcta attaaggttt gccctgcctg cagcctcccc cacaaataat 38520

gaacagcaga aagactggac ggggaaacct atcaatcctg cccccagcca tggtgaggaa 38580

gccccaagcc atggtgacac acagcagcac tgcagatagc cagacacatg gctatcctag 38640

agaggctggc aaggagttcg tggctgcaaa agaagtttct ggagcaagag agagctcgct 38700

cttgggagtc aggacctccg gggagagcag agggttccga cggattcctt tatgagtcag 38760

tctctctctc ccttttaaat ggtgggaacc ctccccaaaa cctttcccca gacacattct 38820

cctgtgcccc tcagagaggc atgtgatgtg caaggaaaat aataggatat aaaacacatc 38880

aagtagaaaa tttcttatac ttc 38903

<210>2

<211>20

<212>DNA

＜213〉homo sapiens (homo sapiens)

<400>2

cttgttggat ctgtgcagtt 20

<210>3

<211>19

<212>DNA

＜213〉homo sapiens (homo sapiens)

<400>3

ttgttggatc tgtgcagtc 19

<210>4

<211>23

<212>DNA

＜213〉homo sapiens (homo sapiens)

<400>4

ccagttctgc cgtccataaa atg 23

<210>5

<211>61

<212>DNA

＜213〉homo sapiens (homo sapiens)

<400>5

cttgttggat ctgtgcagty tgagaagaat gatatgccca ttttatggac ggcagaactg 60

g 61

Claims (10)

1. method that the diabetes B susceptibility of individuality is diagnosed is characterized in that it comprises step:

Detect this individual GLP1R gene, transcript and/or albumen, and compare with normal GLP1R gene, transcript and/or albumen,

The possibility that there are differences with regard to showing this individuality trouble diabetes B is higher than normal population.

2. the method for claim 1 is characterized in that, detection be gene or the transcript of GLP1R, and with normal GLP1R nucleotide sequence comparing difference.

3. the method for claim 1 is characterized in that, described difference is the single nucleotide polymorphism that is selected from down group:

The genotype in 4347 GA and this site is AA;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

4. whether a test sample exists the method for the single nucleotide polymorphism of GLP1R gene, it is characterized in that, comprises step:

(a) with the GLP1R gene of GLP1R gene-specific primer amplification sample, obtain amplified production; With

(b) detect whether there is the single nucleotide polymorphism that is selected from down group in the amplified production:

The genotype in 4347 G A and this site is AA;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

5. method as claimed in claim 4 is characterized in that, described method also comprises step: the sample supplier carried out the BMI index analysis.

6. the test kit of a complementary detection diabetes B is characterized in that, it comprises the primer and the specification sheets of specific amplification GLP1R gene or transcript.

7. test kit as claimed in claim 6 is characterized in that, it also contains the reagent that is selected from down group:

(a) with mutable site bonded probe;

(b) restriction enzyme in identification mutational site.

8. test kit as claimed in claim 7 is characterized in that, described sudden change is the single nucleotide polymorphism that is selected from down group:

4347 G A;

Wherein, the nucleotide position numbering is based on SEQ ID NO:1.

9. test kit as claimed in claim 6 is characterized in that, described primer is the primer of sequence shown in SEQ IDNO:2,3 and 4.

10. a GLP1R gene and proteic purposes is characterized in that, are used to prepare the reagent of complementary detection diabetes B.

Images