WO2007077422A2 - Allele-specific sequencing - Google Patents

Allele-specific sequencing Download PDF

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WO2007077422A2
WO2007077422A2 PCT/GB2006/004904 GB2006004904W WO2007077422A2 WO 2007077422 A2 WO2007077422 A2 WO 2007077422A2 GB 2006004904 W GB2006004904 W GB 2006004904W WO 2007077422 A2 WO2007077422 A2 WO 2007077422A2
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haplotype
gene
hypertension
stroke
nucleic acid
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French (fr)
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WO2007077422A3 (en
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David Neil Cooper
Martin Horan
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University College Cardiff Consultants Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the invention relates to a novel method for identifying at least one polymorphism or mutation in a given gene and also the corresponding haplotype, particularly, but not exclusively, in the growth hormone gene.
  • the invention also relates to a kit for undertaking the aforementioned method, including parts thereof. Further, the invention relates to novel haplotypes in the promoter region of the growth hormone gene and the use of these haplotypes in diagnosing hypertension or stroke.
  • the gene encoding pituitary growth hormone (GH 1) is located on chromosome 17q23 within a cluster of five related genes ( Figure 1). This 66.5 kb cluster has now been sequenced in its entirety [Chen et al. Genomics 4 479-497 (1989) and see Figure 3].
  • the other loci present in the growth hormone gene cluster are two chorionic somatomammotropin genes ⁇ CSH1 and CSH2), a chorionic somatomammotropin pseudogene ⁇ CSHP1) and a growth hormone gene (GH2). These genes are separated by intergenic regions of 6 to 13 kb in length, lie in the same transcriptional orientation, are placentally expressed and are under the control of a downstream tissue-specific enhancer.
  • the GH2 locus encodes a protein that differs from the GH7-derived growth hormone at 13 amino acid residues. All five genes share a very similar structure with five exons interrupted at identical positions by short introns, 260bp, 209bp, 92bp and 253bp in length in the case of GH 1 ( Figure 2).
  • Exon 1 of the GH 1 gene contains 60bp of 5 ! untranslated sequence (although an alternative transcriptional initiation site is present at -54), codons -26 to -24 and the first nucleotide of codon -23 corresponding to the start of the 26 amino acid leader sequence.
  • Exon 2 encodes the rest of the leader peptide and the first 31 amino acids of mature GH.
  • Exons 3-5 encode amino acids 32-71 , 72-126 and 127-191 , respectively.
  • Exon 5 also encodes 112bp 3 1 untranslated sequence culminating in the polyadenylation site.
  • An AIu repetitive sequence element is present 100bp 3' to the GH 1 polyadenylation site. Although the five related genes are highly homologous throughout their 5' flanking and coding regions, they diverge in their 3' flanking regions.
  • polymorphisms/mutations in the GH1 gene can lead to Metabolic Syndrome and so a knowledge of GH1 haplotype is important in predicting and diagnosing this further clinical condition.
  • Hypertension or high blood pressure
  • primary hypertension the arterial blood pressure persistently exceeds 150/90mmhg and is generally acknowledged to be of unknown causation although diet, stress and life style are thought to have a part play.
  • Secondary hypertension is usually a symptom of an underlying condition and is most commonly due to renal disease. However, it may also be caused by phaeochromocytona, by excess secretion of glucocorticoids or of aldosterone, or by coarctation of the aorta.
  • a stroke or a cerebrovascular accident occurs when the blood supply to a part of the brain is suddenly interrupted by an occlusion, haemorrhaging or some other means. Interruption of blood supply by occlusion is the most common form of stroke, occurring in 90% of cases, and is termed an ischemic stroke. In contrast, a haemorrhagic stroke accounts for less than 10% of all strokes. Temporary or permanent loss of blood supply to the brain can result in cell death leaving a part of the brain no longer being able to function.
  • the most common underlying cause of a stroke is a blood clot that lodges in an artery and blocks the supply of blood flow to the brain. Blood clots can form as a result of poor diet, lack of exercise, hypertension and stress. There has been no suggestion in the prior art that stroke may be linked to growth hormone activity.
  • the invention has application in determining the characteristics of any given gene with a view to identifying, typically, determinative polymorphisms, mutations or haplotypes.
  • a method of allele-specific sequencing comprising the use of at least one allele-specific primer targeted to at least one known SNP site within a nucleic acid molecule wherein hybridisation of the primer and the subsequent transcription of a complementary nucleic acid strand produces a nucleic acid strand that can be sequenced, from a known SNP start site, with a view to identifying the existence of other, known SNPs within the transcribed product.
  • a method of allele-specific sequencing comprising: (a) obtaining a test sample comprising a nucleic acid molecule encoding the
  • GH 1 gene or at least the promoter thereof
  • nucleic acid molecule (b) exposing said nucleic acid molecule to at least one allele-specific primer which primer is adapted to bind, at its last 3' base, to at least one selected SNP site within said gene or said proximal promoter; (c) providing suitable conditions for transcription to take place whereby a complementary strand of said nucleic acid is produced from said selected SNP site onwards;
  • a plurality of primers may be used.
  • the distance between SNP sites and the number of SNP variants may determine the number of primers that need to be used. For example, where there are a number of variants within a relatively short distance, i.e. approximately 30 bases, then more than one allele-specific sequence may be required.
  • an allele-specific primer for use in the aforementioned methodology.
  • an allele-specific primer comprising an oligonucleotide complementary to a region of a gene containing a selected SNP site wherein the 3' base of the oligonucleotide is complementary to the SNP.
  • the 3' base is used for the identification of the SNP and acts as a start-site marker for the ailele-specific sequencing.
  • allele-specific sequencing relies upon a knowledge of the SNPs to be identified.
  • an allele-specific primer will be selected so as to hybridise to a region of a gene upstream of a number of SNP sites whereby following binding of the 3' base of the allele-specific primer to its selected SNP suitable conditions can be provided for conventional transcription, thus resulting in the production of a strand of nucleic acid complementary to the gene under investigation.
  • This complementary strand whose start site is known by virtue of the SNP identified by the 3' base of the primer, enables the transcribed nucleic acid to be sequenced and investigated for downstream SNPs.
  • allele-specific sequencing has been used to investigate the growth hormone gene (GH1) and therefore the primers described herein are specific for SNPs in this gene and in particular the proximal promoter region of the growth hormone gene and specifically SNPs located at -308, -278, -75, -57, -6, -1 and +59 having regard to the transcriptional start site.
  • Suitable primers for use in one aspect of the invention are therefore as follows.
  • Oligonucleotide primer sequences for GH1 allele-specific sequencing are provided.
  • the last, 3'-most base is SNP allele-specific; a 3' mismatch is given in lower case.
  • TSS transcriptional start site
  • primers for use with different genes and designed to hybridise to a selected SNP therein can be designed with a view to working the invention in respect of any selected gene.
  • at least one core haplotype as indicated in Table 2, for the purpose of assessing whether a person is susceptible to hypertension or stroke.
  • the core haplotypes referred to in Table 2 comprise the nucleic acid bases at SNP sites -476, -278, -168, -57, -6 and +16.
  • the haplotype used for performing the above assessment comprises one of the "at risk” haplotypes indicated in Table 2 and so, for the purpose of performing an assessment relating to hypertension the haplotypes comprise CH3, CH7, CH8, CH9, CH10, CH11 and CH12, and, in the case of performing an assessment relating to stroke the "at risk” haplotypes comprise any one of the following CH2, CH7, CH9, CH10, CH11. CH13, CH15 or CH16.
  • a method for determining the existence of, or susceptibility to, hypertension comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from hypertension:
  • a method for determining the existence of, or susceptibility to, hypertension comprising:
  • the individual is female.
  • the preferred haplotypes are CH3, CH8 or CH 12.
  • a method for determining the existence of, or susceptibility to, stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from stroke:
  • a method for determining the existence of, or susceptibility to, stroke comprising:
  • the individual is female.
  • the preferred haplotypes are CH2, CH13, CH15 or CH16.
  • haplotypes CH7, CH9, CH10 and CH11 associate with hypertension or stroke, it follows that, in respect of hypertension, haplotypes CH3, CH8 and CH12 are particularly discriminatory for hypertension; and, in respect of stroke, haplotypes CH2, CH13, CH15 and CH16 are particularly discriminatory for stroke.
  • a novel haplotype comprising a haplotype selected from the following group:
  • a method for determining the existence of, or susceptibility to, hypertension or stroke comprising:
  • a method for determining the existence of, or susceptibility to, hypertension or stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNPs sites: -476, -268, -168, -57, -6 and +16 and, where any one of the following haplotypes exist determining that the individual is at risk from hypertension or stroke:
  • Study 1 A total of 2,886 healthy adults (1630 males, mean age 44.9+/-22.4 years; 1256 females, mean age 39.4+/-19.5 years) were selected at random from the general population and were studied as part of an ongoing Anglo-shire Collaboration Trial (ACCT) (McEniery et al., 2005). Individuals with diabetes, hypercholesterolemia (serum cholesterol >6.5 mmol/L) or a history of
  • cardiovascular disease defined as a clinical history or evidence on examination
  • Blood pressure, arterial stiffness and serum markers were measured and reported smoking status was noted.
  • Study 2 111 consecutive hypertensive patients (58 males, 53 females; mean age 60.0+/-15.1 years, range 19-83) were recruited from Addenbrooke's Hospital, Cambridge. Blood pressure, arterial stiffness and serum markers were measured and reported smoking status was noted. A total of 121 controls (59 males, 62 females) were also selected so as to match the patients for gender, age (mean age 60.9+/-13.3 years, range 19-83) and smoking status. Patients were recruited from Addenbrooke's Hospital, Cambridge; secondary causes of hypertension were excluded and fewer than 10% of patients were on anti- hypertensive treatment.
  • Study 3 155 stroke patients of Caucasian origin (73 males, 78 females, 4 gender not recorded; 31 smokers, 116 non-smokers, 8 smoking status not recorded; mean age 72.1 ⁇ 11.7 years) were recruited from the Nottingham Stroke service. A total of 158 local controls were matched to the cases for age, gender and smoking status (76 males, 82 females; 35 smokers, 123 non- smokers; mean age 71.8 ⁇ 3.5 years). These individuals were selected from a larger group recruited for a previous study (Britton et al., 1994). Haemodynamic measurements
  • Peripheral blood pressure was recorded in the brachial artery of the dominant arm using a validated oscillometric technique (HEM-705CP; Omron Corporation, Japan; O'Brien et al. 1996).
  • Radial artery pressure waveforms were obtained with a high fidelity micromanometer (SPC-301 ; Millar Instruments, Texas, USA) from the wrist, and from this, a corresponding central arterial pressure waveform was generated using a validated transfer function (Sphygmocor; AtCor Medical, Sydney, Australia; Karamonoglu et al. 1993, Segers et al. 2001 , Pauca et al. 2001 ) as previously described (Wilkinson et al., 1998).
  • the augmentation index (AIx) a composite measure of systemic arterial stiffness, wave reflection and heart rate, was determined using the integral software (Safar et al., 2000; Rashid et al., 2003).
  • the aortic pulse wave velocity (PVW) was measured using the same device by sequentially recording ECG-gated carotid and femoral artery pressure waveforms (Wilkinson et al., 1998), and brachial PWV from carotid and radial arteries (Wilkinson et al., 1998). All measurements were performed in duplicate, and the averages of the two values were used in the subsequent analysis.
  • Genomic DNA was extracted from venous blood using standard methods. Genotyping was performed in study 2 and 3 subjects using published primers as described below.
  • 3.2 kb fragments specific for the GH1 gene were PCR-amplified from every patient and control individual. The entire coding region, introns, promoter and 3'untranslated region were then directly sequenced on an Applied Biosystems 3100 DNA Genetic Analyzer as previously described (Horan et al., 2003; Millar et al., 2003). Around 20% of individuals tested were found to be homozygous for all 15 promoter SNPs under study, in which case the identity of the promoter haplotypes was clear from the outset. For heterozygotes, the 3.2 kb GH1 gene fragments were cloned and one clone was sequenced to identify unambiguously the two GH1 promoter haplotypes.
  • GH 1 promoter haplotype identification using allele-specific sequencing (ASS) PCR amplification and sequencing of a 3.2 kb GH1 gene-specific fragment were performed as described above.
  • Haplotypes were identified by allele-specific sequencing (ASS) of the GH1 proximal promoter region using a BigDye
  • Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and an ABI 3100 DNA Genetic Analyser. Sequencing reactions were performed as follows: 96 0 C 30 sec, 62 0 C 4 min for 30 cycles. Oligonucleotide primers used for ASS were designed so as to include bases immediately upstream or downstream of a SNP site, with the 3'-most base being allele-specific (Table 1).
  • ASS The efficiency of ASS was determined by the blind analysis of 50 control DNA samples. PCR and sequencing were performed in order to identify the genotype of each sample. The same PCR fragments were then subjected to (i) cloning and sequencing and (ii) ASS in both 5' and 3' directions to yield promoter haplotypes from the 100 alleles. Perfect concordance of haplotype identification was achieved but ASS proved to be considerably faster than traditional cloning and sequencing. Indeed, ASS virtually eliminates the need for cloning. Thus validated, ASS was used to determine the GH 1 promoter haplotypes of stroke patients and the matched controls.
  • GH1 promoter haplotype identification was performed by means of the novel technique of ASS using seven discriminant SNPs from the 16 previously identified SNPs (Giordano et al., 1997; Wagner et al., 1997). Direct sequencing of the original 3.2kb PCR product initially allowed identification of the combined genotype (both alleles), and this information was used to select ASS primers to potentiate GH1 allele discrimination. Original PCR products were then re- sequenced using appropriate allele-specific primers targeted to SNP positions - 308G, -278G, -75G, -57G, -6A, -1T and +59T (Table 1 ). Use of these primers allowed reproducible haplotype identification.
  • ASS could not be used to derive haplotypes owing to the close proximity of these SNPs to each other.
  • the initial PCR product had to be cloned and sequenced in order to identify the promoter haplotypes.
  • SNP variants occurring between positions -6 and +25 were only found to occur at a frequency of -1-5%.
  • the application of ASS methodology in its current form appears capable of successfully identifying a minimum of 95% of all observed GH 1 promoter haplotypes.
  • GH1 promoter haplotyping for 97 hypertensive patients and 112 matched controls, and for 154 stroke patients and 152 matched controls, was performed as described by Horan et al. (2003). A small number of samples were found to be refractory to PCR amplification and were therefore excluded. Since in excess of 100 GH1 promoter haplotypes have been identified to date (unpublished data), we opted to consider only the 'core haplotypes 1 comprising 17 different combinations of the six 'key SNPs' identified by Horan et al. (2003) as major determinants of GH1 gene expression.
  • those core haplotypes with an odds ratio higher than that of the modal haplotype H1 were classified as 'at risk' (see Table 2).
  • GH 1 core promoter haplotypes were found to differ significantly not only between hypertensive patients and controls but also between stroke patients and controls. Further, four core promoter haplotypes (CH7, CH9, CH10 and
  • CH11 CH11 were consistently classified as 'at risk' for both hypertension and stroke patients. Intriguingly, the association between GH1 promoter haplotype and the risk of hypertension and stroke was much greater in females than in males.
  • the waist-to-hip ratio correlates strongly and inversely with HDL cholesterol levels in hypopituitarism (Abdu et al., 2001 ), a finding which suggests that the excess predicted vascular risk in growth hormone-deficient hypopituitary females may be largely an indirect reflection of the greater increase in central adiposity in women than in men. The metabolic and vascular consequences of growth hormone deficiency would therefore appear to be greater in females than in males. The gender-specific GH1 haplotype associations with hypertension and stroke reported here are broadly consistent with this conclusion. It is plausible to speculate that interactions with sex steroids may account for these differences. Indeed, Growth Hormone replacement therapy in females also receiving sex steroids is dependent upon the route of administration of the sex steroids, viz oral or via a skin patch.
  • RH risk haplotype classification (+:yes, o: no); a: order of SNPs as in Table 4 of Horan et al. (2003); n.a.: not applicable. References
  • GH1 gene expression is influenced in a complex haplotype-dependent fashion by polymorphic variation in both the proximal promoter and the locus control region.
  • Pauca AL O'Rourke MF, Kon ND. (2001 ) Prospective evaluation of a method for estimating ascending aortic pressure from the radial artery pressure waveform.

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Abstract

The invention relates to a novel method of allele-specific sequencing in any gene which involves the knowledge of the existing SNPs within said gene and, in particular, those SNPs that are phenotypically significant for a given condition.

Description

ALLELE-SPECIFIC SEQUENCING
The invention relates to a novel method for identifying at least one polymorphism or mutation in a given gene and also the corresponding haplotype, particularly, but not exclusively, in the growth hormone gene. The invention also relates to a kit for undertaking the aforementioned method, including parts thereof. Further, the invention relates to novel haplotypes in the promoter region of the growth hormone gene and the use of these haplotypes in diagnosing hypertension or stroke.
Human stature is a highly complex trait resulting from the interaction of multiple genetic and environmental factors. Since familial short stature is already known to be associated with inherited mutations of the growth hormone (GH1) gene, it appears reasonable to suppose that polymorphic variation in this pituitary- expressed gene can also influence adult height.
The gene encoding pituitary growth hormone (GH 1) is located on chromosome 17q23 within a cluster of five related genes (Figure 1). This 66.5 kb cluster has now been sequenced in its entirety [Chen et al. Genomics 4 479-497 (1989) and see Figure 3]. The other loci present in the growth hormone gene cluster are two chorionic somatomammotropin genes {CSH1 and CSH2), a chorionic somatomammotropin pseudogene {CSHP1) and a growth hormone gene (GH2). These genes are separated by intergenic regions of 6 to 13 kb in length, lie in the same transcriptional orientation, are placentally expressed and are under the control of a downstream tissue-specific enhancer. The GH2 locus encodes a protein that differs from the GH7-derived growth hormone at 13 amino acid residues. All five genes share a very similar structure with five exons interrupted at identical positions by short introns, 260bp, 209bp, 92bp and 253bp in length in the case of GH 1 (Figure 2).
Exon 1 of the GH 1 gene contains 60bp of 5! untranslated sequence (although an alternative transcriptional initiation site is present at -54), codons -26 to -24 and the first nucleotide of codon -23 corresponding to the start of the 26 amino acid leader sequence. Exon 2 encodes the rest of the leader peptide and the first 31 amino acids of mature GH. Exons 3-5 encode amino acids 32-71 , 72-126 and 127-191 , respectively. Exon 5 also encodes 112bp 31 untranslated sequence culminating in the polyadenylation site. An AIu repetitive sequence element is present 100bp 3' to the GH 1 polyadenylation site. Although the five related genes are highly homologous throughout their 5' flanking and coding regions, they diverge in their 3' flanking regions.
A number of investigations have been undertaken on the GH1 gene and as a result of same known polymorphisms in the human GH1 gene promoter/51 of the five untranslated regions have been identified and are as detailed in our co- pending patent application VVO 03/042245. Additionally, other investigations have documented gross deletions in the GH1 gene, micro deletions in the GH1 gene and single base pair substitutions. All these variants of the GH 1 gene are documented in our co-pending patent application VVO 03/042245 and the skilled reader is therefore referred to this patent specification for more background information concerning the nature of GH1 variants that exist.
Additionally, in our co-pending application WO 04/057028 we describe how variation at the 15 of the 16 known SNP locations within the proximal promoter of the GH1 gene manifest as a total of 40 different promoter haplotypes. Also in this document, we demonstrated that 6 of the SNPs act as major determinants of GH1 gene expression, whilst a further 6 SNPs are only marginally informative of GH 1 gene expression.
Given the genetic complexity of human stature, our data have led us to conclude that certain combinations of SNP's, and so haplotypes, can have significantly determinative effects on human stature. Accordingly, knowledge of this information is useful for identifying individuals who suffer from under-expression of growth hormone and so require replacement therapy at least until puberty.
Additionally, polymorphisms/mutations in the GH1 gene can lead to Metabolic Syndrome and so a knowledge of GH1 haplotype is important in predicting and diagnosing this further clinical condition.
Hypertension, or high blood pressure, is classified into one of two types; primary or secondary hypertension. In primary hypertension the arterial blood pressure persistently exceeds 150/90mmhg and is generally acknowledged to be of unknown causation although diet, stress and life style are thought to have a part play. Secondary hypertension, as the name implies, is usually a symptom of an underlying condition and is most commonly due to renal disease. However, it may also be caused by phaeochromocytona, by excess secretion of glucocorticoids or of aldosterone, or by coarctation of the aorta.
As will be apparent, in cases of primary or secondary hypertension either the causal factors are unknown or well documented, but in either case there has been no link to growth hormone.
A stroke or a cerebrovascular accident (CVA) occurs when the blood supply to a part of the brain is suddenly interrupted by an occlusion, haemorrhaging or some other means. Interruption of blood supply by occlusion is the most common form of stroke, occurring in 90% of cases, and is termed an ischemic stroke. In contrast, a haemorrhagic stroke accounts for less than 10% of all strokes. Temporary or permanent loss of blood supply to the brain can result in cell death leaving a part of the brain no longer being able to function. The most common underlying cause of a stroke is a blood clot that lodges in an artery and blocks the supply of blood flow to the brain. Blood clots can form as a result of poor diet, lack of exercise, hypertension and stress. There has been no suggestion in the prior art that stroke may be linked to growth hormone activity.
As a result of our investigations we have, somewhat surprisingly, discovered that mutations/polymorphisms in the growth hormone gene can be identified in individuals suffering from either hypertension and stroke. Indeed, as a result of our investigations we have identified novel and significant haplotypes that have implications for hypertension and stroke. We consider that the identification of our novel haplotypes indicates that GH screening is being under-used due to a lack of knowledge of the relevance of mutations/polymorphisms in the GH1 gene.
Accordingly, we have developed a novel methodology for allele-specific sequencing which enables us to determine the number and nature of SNPs in a given nucleic acid molecule and so characterise the haplotype corresponding thereto. This information has allowed us to determine the nature of haplotypes that are indicative of a given condition.
The invention has application in determining the characteristics of any given gene with a view to identifying, typically, determinative polymorphisms, mutations or haplotypes.
In the following description of the invention we have used our novel technique with particular reference to the growth hormone gene. However, those skilled in the art will appreciate that this technique can be applied to any given gene provided at least one SNP, ideally one that is determinative of a given characteristic to be investigated, is known. Statement of Invention
According to the invention there is therefore provided a method of allele-specific sequencing comprising:
(a) obtaining a test sample comprising a nucleic acid molecule to be sequenced;
(b) exposing said nucleic acid molecule to at least one allele-specific primer which is adapted to bind, at its last 3' base, to at least one selected SNP site within said molecule;
(c) providing suitable conditions for transcription to take place whereby a complementary strand of said nucleic acid is produced from said selected SNP site onwards;
(d) sequencing said complementary strand in order to determine the existence of other known SNPs; and, optionally,
(e) using this sequence information to determine the haplotype of said individual.
According to the invention there is therefore provided a method of allele-specific sequencing comprising the use of at least one allele-specific primer targeted to at least one known SNP site within a nucleic acid molecule wherein hybridisation of the primer and the subsequent transcription of a complementary nucleic acid strand produces a nucleic acid strand that can be sequenced, from a known SNP start site, with a view to identifying the existence of other, known SNPs within the transcribed product.
According to a further aspect of the invention there is provided a method of allele-specific sequencing comprising: (a) obtaining a test sample comprising a nucleic acid molecule encoding the
GH 1 gene, or at least the promoter thereof;
(b) exposing said nucleic acid molecule to at least one allele-specific primer which primer is adapted to bind, at its last 3' base, to at least one selected SNP site within said gene or said proximal promoter; (c) providing suitable conditions for transcription to take place whereby a complementary strand of said nucleic acid is produced from said selected SNP site onwards;
(d) sequencing said complementary strand in order to determine the existence of other known SNPs; and, optionally, (e) using this sequence information to determine the haplotype of said individual.
In a preferred embodiment of the invention a plurality of primers may be used. Those skilled in the art will appreciate that the distance between SNP sites and the number of SNP variants may determine the number of primers that need to be used. For example, where there are a number of variants within a relatively short distance, i.e. approximately 30 bases, then more than one allele-specific sequence may be required. According to a further aspect of the invention there is provided an allele-specific primer for use in the aforementioned methodology.
According to a further aspect of the invention there is provided an allele-specific primer comprising an oligonucleotide complementary to a region of a gene containing a selected SNP site wherein the 3' base of the oligonucleotide is complementary to the SNP. Those skilled in the art will appreciate that the 3' base is used for the identification of the SNP and acts as a start-site marker for the ailele-specific sequencing.
In summary, as will be apparent to those skilled in the art, allele-specific sequencing according to the invention relies upon a knowledge of the SNPs to be identified. Most suitably, an allele-specific primer will be selected so as to hybridise to a region of a gene upstream of a number of SNP sites whereby following binding of the 3' base of the allele-specific primer to its selected SNP suitable conditions can be provided for conventional transcription, thus resulting in the production of a strand of nucleic acid complementary to the gene under investigation. This complementary strand, whose start site is known by virtue of the SNP identified by the 3' base of the primer, enables the transcribed nucleic acid to be sequenced and investigated for downstream SNPs.
In the methodology described herein allele-specific sequencing has been used to investigate the growth hormone gene (GH1) and therefore the primers described herein are specific for SNPs in this gene and in particular the proximal promoter region of the growth hormone gene and specifically SNPs located at -308, -278, -75, -57, -6, -1 and +59 having regard to the transcriptional start site.
Suitable primers for use in one aspect of the invention are therefore as follows.
TABLE 1
Oligonucleotide primer sequences for GH1 allele-specific sequencing.
Figure imgf000010_0001
The last, 3'-most base is SNP allele-specific; a 3' mismatch is given in lower case. TSS (transcriptional start site).
However, those skilled in the art will appreciate that primers for use with different genes and designed to hybridise to a selected SNP therein can be designed with a view to working the invention in respect of any selected gene. According to a yet further aspect of the invention there is provided at least one core haplotype, as indicated in Table 2, for the purpose of assessing whether a person is susceptible to hypertension or stroke.
The core haplotypes referred to in Table 2 comprise the nucleic acid bases at SNP sites -476, -278, -168, -57, -6 and +16.
In a preferred embodiment of the invention the haplotype used for performing the above assessment comprises one of the "at risk" haplotypes indicated in Table 2 and so, for the purpose of performing an assessment relating to hypertension the haplotypes comprise CH3, CH7, CH8, CH9, CH10, CH11 and CH12, and, in the case of performing an assessment relating to stroke the "at risk" haplotypes comprise any one of the following CH2, CH7, CH9, CH10, CH11. CH13, CH15 or CH16.
According to a yet further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, hypertension comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from hypertension:
Figure imgf000011_0001
Figure imgf000012_0001
According to a yet further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, hypertension comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least a proximal promoter region of the growth hormone gene {GH1) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000012_0002
and; (d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, hypertension.
In a preferred embodiment of the invention the individual is female.
According to a yet further preferred method of either aspect of the invention relating to a method for determining the existence of, or susceptibility to, hypertension the preferred haplotypes are CH3, CH8 or CH 12.
According to a yet further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from stroke:
Figure imgf000013_0001
According to a yet further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, stroke comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least a proximal promoter region of the growth hormone gene (GH1) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000014_0001
and;
(d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, stroke.
In a preferred embodiment of the invention the individual is female. In a preferred method of either aspect of the invention relating to a method for determining the existence of, or susceptibility to, stroke the preferred haplotypes are CH2, CH13, CH15 or CH16.
According to a yet further aspect of the invention there is provided the use of any one or more of the following core haplotypes:
Figure imgf000015_0001
in the diagnosis or treatment of hypertension or stroke.
Those skilled in the art will appreciate that since haplotypes CH7, CH9, CH10 and CH11 associate with hypertension or stroke, it follows that, in respect of hypertension, haplotypes CH3, CH8 and CH12 are particularly discriminatory for hypertension; and, in respect of stroke, haplotypes CH2, CH13, CH15 and CH16 are particularly discriminatory for stroke.
According to a yet further aspect of the invention there is provided, in the GH1 gene, a novel haplotype comprising a haplotype selected from the following group:
Figure imgf000016_0001
According to a yet further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, hypertension or stroke comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least the proximal promoter region of the growth hormone gene (GH7) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000016_0002
and;
(d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, hypertension or stroke. According to a further aspect of the invention there is provided a method for determining the existence of, or susceptibility to, hypertension or stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNPs sites: -476, -268, -168, -57, -6 and +16 and, where any one of the following haplotypes exist determining that the individual is at risk from hypertension or stroke:
Figure imgf000017_0001
An embodiment of the invention will now be described by way of example only with reference to the following materials and methods.
Subjects
All subjects were Caucasians. Approval for the studies was obtained from the Local Regional Ethics Committees and written informed consent obtained from each participant.
Study 1: A total of 2,886 healthy adults (1630 males, mean age 44.9+/-22.4 years; 1256 females, mean age 39.4+/-19.5 years) were selected at random from the general population and were studied as part of an ongoing Anglo- Cardiff Collaboration Trial (ACCT) (McEniery et al., 2005). Individuals with diabetes, hypercholesterolemia (serum cholesterol >6.5 mmol/L) or a history of
cardiovascular disease (defined as a clinical history or evidence on examination) were excluded from the analysis, as were subjects receiving any cardiovascular medication. Blood pressure, arterial stiffness and serum markers were measured and reported smoking status was noted.
Study 2: 111 consecutive hypertensive patients (58 males, 53 females; mean age 60.0+/-15.1 years, range 19-83) were recruited from Addenbrooke's Hospital, Cambridge. Blood pressure, arterial stiffness and serum markers were measured and reported smoking status was noted. A total of 121 controls (59 males, 62 females) were also selected so as to match the patients for gender, age (mean age 60.9+/-13.3 years, range 19-83) and smoking status. Patients were recruited from Addenbrooke's Hospital, Cambridge; secondary causes of hypertension were excluded and fewer than 10% of patients were on anti- hypertensive treatment.
Study 3: 155 stroke patients of Caucasian origin (73 males, 78 females, 4 gender not recorded; 31 smokers, 116 non-smokers, 8 smoking status not recorded; mean age 72.1 ±11.7 years) were recruited from the Nottingham Stroke service. A total of 158 local controls were matched to the cases for age, gender and smoking status (76 males, 82 females; 35 smokers, 123 non- smokers; mean age 71.8±3.5 years). These individuals were selected from a larger group recruited for a previous study (Britton et al., 1994). Haemodynamic measurements
Peripheral blood pressure was recorded in the brachial artery of the dominant arm using a validated oscillometric technique (HEM-705CP; Omron Corporation, Japan; O'Brien et al. 1996). Radial artery pressure waveforms were obtained with a high fidelity micromanometer (SPC-301 ; Millar Instruments, Texas, USA) from the wrist, and from this, a corresponding central arterial pressure waveform was generated using a validated transfer function (Sphygmocor; AtCor Medical, Sydney, Australia; Karamonoglu et al. 1993, Segers et al. 2001 , Pauca et al. 2001 ) as previously described (Wilkinson et al., 1998). The augmentation index (AIx), a composite measure of systemic arterial stiffness, wave reflection and heart rate, was determined using the integral software (Safar et al., 2000; Rashid et al., 2003). The aortic pulse wave velocity (PVW) was measured using the same device by sequentially recording ECG-gated carotid and femoral artery pressure waveforms (Wilkinson et al., 1998), and brachial PWV from carotid and radial arteries (Wilkinson et al., 1998). All measurements were performed in duplicate, and the averages of the two values were used in the subsequent analysis.
Laboratory measurements Total serum cholesterol, triglycerides, glucose, glycosylated haemoglobin and C- reactive protein were determined using standard methodology in an accredited laboratory. Genetic analysis
Genomic DNA was extracted from venous blood using standard methods. Genotyping was performed in study 2 and 3 subjects using published primers as described below.
Detection of polymorphisms within the GH 1 gene
3.2 kb fragments specific for the GH1 gene were PCR-amplified from every patient and control individual. The entire coding region, introns, promoter and 3'untranslated region were then directly sequenced on an Applied Biosystems 3100 DNA Genetic Analyzer as previously described (Horan et al., 2003; Millar et al., 2003). Around 20% of individuals tested were found to be homozygous for all 15 promoter SNPs under study, in which case the identity of the promoter haplotypes was clear from the outset. For heterozygotes, the 3.2 kb GH1 gene fragments were cloned and one clone was sequenced to identify unambiguously the two GH1 promoter haplotypes.
GH 1 promoter haplotype identification using allele-specific sequencing (ASS) PCR amplification and sequencing of a 3.2 kb GH1 gene-specific fragment were performed as described above. Haplotypes were identified by allele-specific sequencing (ASS) of the GH1 proximal promoter region using a BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) and an ABI 3100 DNA Genetic Analyser. Sequencing reactions were performed as follows: 960C 30 sec, 620C 4 min for 30 cycles. Oligonucleotide primers used for ASS were designed so as to include bases immediately upstream or downstream of a SNP site, with the 3'-most base being allele-specific (Table 1).
The efficiency of ASS was determined by the blind analysis of 50 control DNA samples. PCR and sequencing were performed in order to identify the genotype of each sample. The same PCR fragments were then subjected to (i) cloning and sequencing and (ii) ASS in both 5' and 3' directions to yield promoter haplotypes from the 100 alleles. Perfect concordance of haplotype identification was achieved but ASS proved to be considerably faster than traditional cloning and sequencing. Indeed, ASS virtually eliminates the need for cloning. Thus validated, ASS was used to determine the GH 1 promoter haplotypes of stroke patients and the matched controls.
GH1 promoter haplotype identification by allele-specific sequencing (ASS)
GH1 promoter haplotype identification was performed by means of the novel technique of ASS using seven discriminant SNPs from the 16 previously identified SNPs (Giordano et al., 1997; Wagner et al., 1997). Direct sequencing of the original 3.2kb PCR product initially allowed identification of the combined genotype (both alleles), and this information was used to select ASS primers to potentiate GH1 allele discrimination. Original PCR products were then re- sequenced using appropriate allele-specific primers targeted to SNP positions - 308G, -278G, -75G, -57G, -6A, -1T and +59T (Table 1 ). Use of these primers allowed reproducible haplotype identification. In most cases, only one primer was required to derive the promoter haplotype. Where genotype analysis revealed SNP variation both upstream (between -476 to -339) and at position - 308 together with variation at positions -75, -57, -6, -1 or +59, two sequencing reactions were required per sample (1 forward and 1 reverse) to derive both haplotypes. Where SNP variation was found only between positions -6 and
+25, ASS could not be used to derive haplotypes owing to the close proximity of these SNPs to each other. In these instances, the initial PCR product had to be cloned and sequenced in order to identify the promoter haplotypes. However, SNP variants occurring between positions -6 and +25 were only found to occur at a frequency of -1-5%. Thus, the application of ASS methodology in its current form appears capable of successfully identifying a minimum of 95% of all observed GH 1 promoter haplotypes.
GH1 promoter haplotyping for 97 hypertensive patients and 112 matched controls, and for 154 stroke patients and 152 matched controls, was performed as described by Horan et al. (2003). A small number of samples were found to be refractory to PCR amplification and were therefore excluded. Since in excess of 100 GH1 promoter haplotypes have been identified to date (unpublished data), we opted to consider only the 'core haplotypes1 comprising 17 different combinations of the six 'key SNPs' identified by Horan et al. (2003) as major determinants of GH1 gene expression. The core haplotypes referred to in the above table comprise the nucleic acid at SNP site -476, -278, -168, -56, -6 and +16. Comparison by means of a permutation test revealed that the observed case and control core haplotype spectra (Table 3) differed significantly from each other in both studies 2 and 3 (hypertension: χ2=18.861 , empirical
P=0.0376; stroke: χ2=23.829, empirical P=0.0273). Inclusion of the putatively
functional intron 4 (1169-T/A) polymorphism (Hasegawa et al., 2000) in the comparison of hypertensives and controls reduced the significance of the
observed haplotype frequency difference (χ2=23.591, empirical P=O.1217), most
probably because the intron 4 1169-T/A polymorphism was not itself associated
with hypertension (genotype-based χ2=1.723, 2 d.f., P=0.423). Stratification of the core haplotype analysis by gender yielded a consistently stronger phenotype
association in females (hypertension: χ2=20.390, empirical P=0.0102; stroke:
χ2=18.500, empirical P=O.1144) than in males (hypertension: χ2=11.751 ,
empirical P=O.1968; stroke: χ2=9.436, empirical P=0.6980).
For the purposes of further analysis, those core haplotypes with an odds ratio higher than that of the modal haplotype H1 , were classified as 'at risk' (see Table 2). In agreement with the gender differences observed at the core haplotype level, the odds ratios (OR) associated with the presence of at least one GH1 promoter risk haplotype were significantly greater than unity in females (hypertension: OR=2.87, 95%CI: 1.21-6.91 ; stroke OR=2.14, 95%CI: 1.07- 4.28), but not in males (hypertension: OR=1.64, 95%CI: 0.68-3.97; stroke OR=1.51 , 95%CI: 0.75-3.05).
Height and central systolic blood pressure (CSBP) measurements were available for the subjects examined in the hypertension study. In keeping with the initial results obtained from the Anglo-Cardiff Collaboration Trial (Study 1 , see above), the two parameters were found to be significantly correlated with one another, both in patients (Spearman rank correlation coefficient r=-0.2736, P=0.0013) and in controls (r—0.1859, P=0.0255). However, this negative correlation was much stronger in individuals carrying at least one GH1 promoter risk haplotype (patients: r=-0.3489, P= 0.0014; controls: r=-0.4751 , PO.0001 ) than in those lacking a risk haplotype (patients: r=-0.1605, P= 0.1356, controls: r=-0.0575, P= 0.3181).
In this study, we have noted an inverse correlation between height and central systolic blood pressure in both hypertensive patients and normal controls (NB. No such correlation was however noted with peripheral systolic blood pressure). GH 1 core promoter haplotypes were found to differ significantly not only between hypertensive patients and controls but also between stroke patients and controls. Further, four core promoter haplotypes (CH7, CH9, CH10 and
CH11 ) were consistently classified as 'at risk' for both hypertension and stroke patients. Intriguingly, the association between GH1 promoter haplotype and the risk of hypertension and stroke was much greater in females than in males.
Growth hormone-deficient hypopituitary women also manifest greater disturbances in body composition than men in comparison to matched controls (Beshyah et al., 1995; Abdu et al., 2001), particularly a greater increase in waist- to-hip ratio and body mass index. The waist-to-hip ratio is a marker of visceral fat accumulation and is associated with increased vascular morbidity and mortality (Larsson et al., 1984). Moreover, the waist-to-hip ratio correlates strongly and inversely with HDL cholesterol levels in hypopituitarism (Abdu et al., 2001 ), a finding which suggests that the excess predicted vascular risk in growth hormone-deficient hypopituitary females may be largely an indirect reflection of the greater increase in central adiposity in women than in men. The metabolic and vascular consequences of growth hormone deficiency would therefore appear to be greater in females than in males. The gender-specific GH1 haplotype associations with hypertension and stroke reported here are broadly consistent with this conclusion. It is tempting to speculate that interactions with sex steroids may account for these differences. Indeed, Growth Hormone replacement therapy in females also receiving sex steroids is dependent upon the route of administration of the sex steroids, viz oral or via a skin patch.
The above mentioned inverse correlation between height and central systolic blood pressure was much stronger in individuals carrying at least one GH1 promoter risk haplotype than in those lacking such a haplotype. It would thus appear that GH1 genotype constitutes a risk factor for hypertension which is either independent of, or interacts with, height. Interestingly, a strong association was found between the presence of at least one GH1 risk haplotype and a family history of stroke at an early age. Since the aetiology of stroke is multifactorial, with a strong albeit heterogeneous heritable component, it would appear very likely that the inheritance of a GH1 risk haplotype accounts for a substantial proportion of familial cases. TABLE 2
Frequency of GH 1 promoter core haplotypes from the two case-control studies.
Figure imgf000026_0001
RH: risk haplotype classification (+:yes, o: no); a: order of SNPs as in Table 4 of Horan et al. (2003); n.a.: not applicable. References
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Hasegawa Y, Fujii K, Yamada M, lgarashi Y, Tachibana K, Tanaka T, Onigata K, Nishi Y, Kato S, Hasegawa T. (2000) Identification of novel human GH-1 gene polymorphisms that are associated with growth hormone secretion and height. J.
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Claims

1. A method of allele-specific sequencing comprising:
(a) obtaining a test sample comprising a nucleic acid molecule to be sequenced;
(b) exposing said nucleic acid molecule to at least one allele-specific primer which is adapted to bind, at its last 3' base, to at least one selected SNP site within said molecule;
(c) providing suitable conditions for transcription to take place whereby a complementary strand of said nucleic acid is produced from said selected SNP site onwards;
(d) sequencing said complementary strand in order to determine the existence of other known SNPs; and, optionally,
(e) using this sequence information to determine the haplotype of said individual.
2. A method of allele-specific sequencing comprising:
(a) obtaining a test sample comprising a nucleic acid molecule encoding the GH 1 gene, or at least the promoter thereof; (b) exposing said nucleic acid molecule to at least one allele-specific primer which primer is adapted to bind, at its last 3J base, to at least one selected SNP site within said gene or said proximal promoter;
(c) providing suitable conditions for transcription to take place whereby a complementary strand of said nucleic acid is produced from said selected SNP site onwards;
(d) sequencing said complementary strand in order to determine the existence of other known SNPs; and, optionally,
(e) using this sequence information to determine the haplotype of said individual.
3. A method according to claim 1 or claim 2 wherein a plurality of primers are used.
4. An allele-specific primer for use in the methodology according to claim 1 or claim 2.
5. A an allele-specific primer comprising an oligonucleotide complementary to a region of a gene containing a selected SNP site wherein the 3' base of the oligonucleotide is complementary to the SNP.
6. An allele-specific primer for use in the method according to claim 1 or 2 comprising an oligonucleotide complementary to a region of said gene containing a selected SNP site wherein the 3' base of the oligonucleotide is complementary to the SNP.
7. An allele-specific primer for use in the method according to claim 2 comprising an oligonucleotide complementary to a region of the proximal promoter region of the growth hormone gene wherein the last 3' base of the oligonucleotide is complementary to any one of the following SNPs: -308, -278, -75, -57, -6, -1 and +59; wherein the numbering is with respect to the transcriptional start site.
8. An allele-specific primer comprising any one of the following oligonucleotides:
CTATCCTGACATCCTTCG
TTGGCCACCATGGCCTGCG
CCCCACCTGTTTCTGaGC
TTCTCTCCCACTGTTGC
CCTTGGGATCCTTGAGCT
GGGCCTTGGGATCCTA
CTTACCTGTAGCCATTGCA
9. Use of a core haplotype, as indicated in Table 2, for assessing the susceptibility to hypertension or stroke.
10. A novel core haplotype in the proximal promoter region of the growth hormone gene comprising any one of the haplotypes listed in Table 2.
11. A method for determining the existence of, or susceptibility to, hypertension comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from hypertension:
Figure imgf000032_0001
12. A method for determining the existence of, or susceptibility to, hypertension comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least a proximal promoter region of the growth hormone gene (GH1) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000032_0002
Figure imgf000033_0001
and;
(d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, hypertension.
13. A method according to claims 11 or 12 wherein the individual is female.
14. A method according to claims 11 -13 wherein the preferred haplotype is one of the following:
Figure imgf000033_0002
15. A method for determining the existence of, or susceptibility to, stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNP sites -476, -278, -168, -57, -6, +16 and, where any one of the following haplotypes exists determining that the individual is at risk from stroke:
Figure imgf000034_0001
16. A method for determining the existence of, or susceptibility to, stroke comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least a proximal promoter region of the growth hormone gene (GH1) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000034_0002
Figure imgf000035_0001
and;
(d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, stroke.
17. A method according to claims 15 or 16 wherein the individual is female.
18. A method according to claims 15-17 wherein the preferred haplotype is one of the following:
Figure imgf000035_0002
19. Use of any one or more of the following core haplotypes:
Figure imgf000035_0003
in the diagnosis or treatment of hypertension or stroke.
20. In the GH 1 gene, a novel haplotype comprising a haplotype selected from the following group:
Figure imgf000036_0001
21. A method for determining the existence of, or susceptibility to, hypertension or stroke comprising:
(a) obtaining a test sample of a nucleic acid molecule encoding at least the proximal promoter region of the growth hormone gene (GH1) from an individual to be tested;
(b) examining said nucleic acid molecule for a plurality of SNPs at the following sites: -476, -278, -168, -57, -6 and +16 in order to determine the corresponding haplotype;
(c) comparing the identified haplotype with any one or more of the following:
Figure imgf000036_0002
and; (d) where the same haplotype is found concluding that the individual may be suffering from, or have a susceptibility to, hypertension or stroke.
22. A method for determining the existence of, or susceptibility to, hypertension or stroke comprising determining the haplotype of the promoter region of the GH1 gene at SNPs sites: -476, -268, -168, -57, -6 and +16 and, where any one of the following haplotypes exist determining that the individual is at risk from hypertension or stroke:
Figure imgf000037_0001
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