WO2007077423A2 - Growth hormone variations - Google Patents

Abstract

The invention relates to novel variants in the growth hormone gene and the corresponding variants in the protein structure and the use of same in the diagnosis of growth hormone dysfunction, hypertension or stroke.

Description

GROWTH HORMONE VARIATIONS

The invention concerns novel growth hormone gene mutations and the corresponding protein variants; a method for screening for the existence of, or a susceptibility to, growth hormone dysfunction involving these naturally occurring growth hormone gene mutations and the corresponding protein variants; moreover, the invention also concerns a kit, including parts thereof, for performing said method. Additionally, the invention concerns the production of novel growth hormone variants, including the means therefor, and the use of said variants as tools in the development of novel therapeutics. Further, the invention concerns the use of the novel GH 1 or GH variants in the screening for, diagnosis or treatment of, hypertension or stroke, including means therefor.

That human stature was influenced by inherited factors was understood more than a century ago. Although familial short stature, with its normally recessive mode of inheritance, was recognised as early as 1912, it was a further quarter century before such families came to be properly documented in the scientific literature. The recognition that recessively inherited short stature was commonly associated with isolated growth hormone (GH) deficiency only came in 1966.

Short stature associated with GH deficiency has been estimated to occur with an incidence of between 1/4000 and 1/10000 live births. Most of these cases are both sporadic and idiopathic, but between 5 and 30% have an affected first- degree relative consistent with a genetic aetiology for the condition. Confirmation of the genetic aetiology of GH deficiency came from the molecular genetic analysis of familial short stature and the early demonstration of mutational lesions in the pituitary-expressed growth hormone (GH1) genes of affected individuals. Familial short stature may also be caused by mutation in a number of other genes {e.g. POU1F1, PR0P1 and GHRHR) and it is important to distinguish these different forms of the condition.

Growth hormone (GH) is a multifunctional hormone that promotes post-natal growth of skeletal and soft tissues through a variety of effects. Controversy remains as to the relative contribution of direct and indirect actions of GH. On one hand, the direct effects of GH have been demonstrated in a variety of tissues and organs, and GH receptors have been documented in a number of cell types. On the other hand, a substantial amount of data indicates that a major portion of the effects of GH are mediated through the actions of GH- dependent insulin-like growth factor I (IGF-I). IGF-1 is produced in many tissues, primarily the liver, and acts through its own receptor to enhance the proliferation and maturation of many tissues, including bone, cartilage, and skeletal muscle. In addition to promoting growth of tissues, GH has also been shown to exert a variety of other biological effects, including lactogenic, diabetogenic, lipolytic and protein anabolic effects, as well as sodium and water retention.

Adequate amounts of GH are needed throughout childhood to maintain normal growth. Newborns with GH deficiency are usually of normal length and weight. Some may have a micropenis or fasting hypoglycemia in conjunction with low linear postnatal growth, which becomes progressively retarded with age. In those with isolated growth hormone deficiency (IGHD), skeletal maturation is usually delayed in association with their height retardation. Truncal obesity, facial appearance younger than expected for their chronological age and delayed secondary dentition are often present. Skin changes similar to those seen in premature ageing may be seen in affected adults.

Familial IGHD comprises several different disorders with characteristic modes of inheritance. Those forms of IGHD known to be associated with defects at the GH 1 gene locus are shown in Table 1 together with the different types of underlying lesion so far detected.

Table 1 : Classification of inherited disorders involving the GH1 gene

The characterisation of these lesions has helped to provide explanations for the differences in clinical severity, mode of inheritance and propensity to antibody formation in response to exogenously administered GH, between these forms of IGHD. Most cases are sporadic and are assumed to arise from cerebral defects that include cerebral oedema, chromosomal anomalies, histiocytosis, infections, radiation, septo-optic dysplasia, trauma, or tumours affecting the hypothalamus or pituitary. Magnetic resonance imaging examinations detect hypothalamic or pituitary anomalies in about 12% of patients who have IGHD.

Although short stature, delayed 'height velocity' or growth velocity, and delayed skeletal maturation are all seen with GH deficiency, none of these is specific for this disorder; other systemic diseases may result in such symptoms. Throughout this specification, 'height velocity' and growth velocity are both to be construed as meaning the rate of change of the subject's or patient's height, such as is measured in centimetres per year.

Stimulation tests to demonstrate GH deficiency use L-Dopa, insulin-induced hypoglycaemia, arginine, insulin-arginine, clonidine, glucagon or propranolol. Inadequate GH peak responses (usually <7-10 ng/mL) differ from test to test.

Testing for concomitant deficiencies of LH, FSH, TSH and ACTH should be performed to determine the extent of pituitary dysfunction and to plan optimal treatment. Recombinant-derived GH is available worldwide and is administered by subcutaneous injection. To obtain an optimal outcome, children with IGHD are usually started on replacement therapy as soon as their diagnosis is established. The initial dosage of recombinant GH is based on body weight or surface area, but the exact amount used and the frequency of administration may vary between different protocols. The dosage increases with increasing body weight to a maximum during puberty. Thereafter, GH treatment should be temporarily discontinued while the individual's GH secretory capacity is re-evaluated. Those with confirmed GH deficiency receive a lower dose of exogenous GH during adult life.

Conditions that are treated with GH include (i) those in which it has proven efficacy and (ii) a variety of others in which its use has been reported but not accepted as standard practice. Disorders in which GH treatment has proven efficacy include GH deficiency, either isolated or in association with combined pituitary hormone deficiency (CPHD) and Turner syndrome. The clinical responses of individuals with the first two disorders to GH replacement therapy varies depending on: (i) the severity of the GH deficiency and its adverse effects on growth, the age at which treatment is begun, weight at birth, current weight and dose of GH; and (ii) recognition and response to treatment of associated deficiencies such as thyroid hormone deficiency; and (iii) whether treatment is complicated by the development of anti-GH antibodies. The outcome of treatment for individuals with Turner syndrome varies with the severity of their short stature, their chromosomal complement, and the age at which treatment was begun.

Additional disorders in which the use of GH has been reported include treatment of certain skeletal dysplasias such as achondroplasia, Prader-Willi syndrome, growth suppression secondary to exogenous steroids or in association with chronic inflammatory diseases such as rheumatoid arthritis, in chronic renal failure, Metabolic Syndrome, extreme idiopathic short stature, Russell-Silver syndrome, SGA (short for gestational age) and intrauterine growth retardation.

The characterisation of familial IGHD at the molecular genetic level is important for several reasons. The identity of the locus involved will indicate not only the likely severity of growth retardation but, more importantly, the appropriateness or otherwise of the various therapeutic regimens now available. Further, detection of the underlying gene lesions serves to confirm the genetic aetiology of the condition. It may also have prognostic value in predicting (i) the severity of growth retardation and (ii) the likelihood of anti-GH antibody formation subsequent to GH treatment. In some instances, knowledge of the pathological lesion(s) can also help to explain an unusual mode of inheritance of the disorder and is therefore essential for the counselling of affected families. Finally, the characterisation of the mutational lesions responsible for cases of IGHD manifesting a dysfunctional (as opposed to a non-functional) GH molecule could yield new insights into GH structure and function. The gene encoding pituitary growth hormone (GH1) 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 GH1 (Figure 2).

Exon 1 of the GH1 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 GH1 polyadenylation site. Although the five related genes are highly homologous throughout their 5' flanking and coding regions, they diverge in their 3' flanking regions. At the cellular level, a single GH molecule binds two GH receptor moles (GHR) causing them to dimerise. Interaction of the dimerised GHR with the intracellular tyrosine protein kinase JAK2 leads to tyrosine phosphorylation of downstream signal transduction molecules, stimulation of mitogen-activated protein (MAP) kinases and induction of signal transducers and activators of transcription (STAT proteins). In this way, GH is able to influence the expression of multiple genes through a number of different signalling pathways.

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/5' of the five untranslated regions have been identified and are as detailed in our co- pending patent application WO 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 GH1 gene are documented in our co-pending patent application WO 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.

Since most cases of familial GH deficiency hitherto described are inherited as an autosomal recessive trait, some examples of the inherited deficiency state are likely to have gone unrecognized owing to small family size. Similarly, cases of GH deficiency resulting from de novo mutations of the GH1 gene could be classified as sporadic, and a genetic explanation for the disorder would neither be entertained nor sought. Finally, depending upon the criteria used for defining the deficiency state, it may be that the full breadth of both the phenotypic and genotypic spectrum of GH deficiency may never have come to clinical attention. For these reasons, current estimates of the prevalence of GH deficiency could be inaccurate and may therefore seriously underestimate the true prevalence in the population.

Typically, mutations and polymorphisms in the GH1 gene are investigated by examining the GH1 gene in individuals who are likely to carry genetic mutations such as those showing mild to severe growth disorders. For example, genetic mutations and polymorphisms are typically identified in individuals showing a height that is below a predicted value at a given age or stage in development. Somewhat unusually, our investigations have led us to examine the growth hormone gene in individuals suffering from either hypertension or stroke.

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 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 three novel and significant variants that have implications for GH diagnosis and treatment. We consider that the identification of our novel variants indicates that GH screening is being under-diagnosed due to a lack of knowledge of relevant mutations in the GH1 gene. Statements of Invention

Accordingly, in a first aspect, the present invention provides an isolated variant of the growth hormone nucleic acid molecule, GH1, comprising one or more of the following substitutions: G>A+447 (Arg16His), T>G+1481 (Phe176Cys) and

T>C+1519 (Cys189Arg).

In particular, the present invention provides a nucleic acid molecule as defined above, wherein the molecule is DNA or RNA, such a cDNA or mRNA.

The present invention also provides a novel transcript of a variant of the GH 1 gene, such as a protein or polypeptide comprising an amino acid sequence encoded by one or more of the aforementioned GH 1 variants.

According to a further aspect of the invention there is therefore provided an isolated polypeptide or protein which is a variant of the growth hormone protein, GH, and which comprises any one or more of the following substitutions: Arg16His Phe176Cys Cys189Arg.

We consider that a knowledge of these novel variants enables us to assay more comprehensively for GH dysfunction and so increases the possibility of IGHD being detected at an early age and so maximises the chances of children with IGHD being identified and started early on replacement therapy so avoiding the clinical disorders referred to above.

Accordingly, the present invention provides a first screening method for screening an individual suspected of having dysfunctional GH which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for any one or more of the following substitutions: G>A+447, T>G+1481 and T>C+1519; and

(d) where one or more of said substitutions exist concluding that the individual suffers from or is likely to suffer from GH dysfunction.

Preferably the test sample comprises genomic DNA or RNA which may be extracted by conventional methods.

According to a further aspect of the invention there is provided a second screening method for screening an individual suspected of having GH dysfunction, which method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein;

(b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below;

(c) examining said sequence for one or more of the following substitutions: Arg16His;

Phe176Cys; or Cys189Arg; and

(d) where one or more of said substitutions exist concluding that the individual suffers from or is likely to suffer from GH dysfunction.

The above screening methods involve an in vitro blood test that can be performed rapidly and efficiently in order to help provide for the early diagnosis of GH dysfunction.

According to a further aspect of the invention there is provided a test kit suitable for carrying out the aforementioned first screening method of the invention which kit comprises:

(a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH 1 gene including one of the following substitutions: G>A+447, T>G+1481 and T>C+1519;

(b) at least one oligonucleotide having a nucleic acid sequence complementary to the wild-type of the gene in one of the regions specified in part (a); and, optionally,

(c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene. Such reagents may include, for example, PCR primers corresponding to the relevant exon of the GH1 gene containing one of the aforementioned substitutions, and/or primers defined herein; and/or other reagents for use in PCR, such as mTaq DNA polymerase.

Ideally, the oligonucleotides or primers in the kit comprise in the range of 15 to 25 bases and, in order to avoid a lack of specificity, ideally, the bases in the oligonucleotides are selected so as to be unique to the region where hybridisation is to take place.

According to a further aspect of the invention there is provided at least one oligonucleotide designed to hybridise to the GH1 gene in order to detect any one or more of the following substitutions: G>A+447, T>G+1481 and T>C+1519.

According to a further aspect of the invention there is provided the use of a GH1 nucleic acid molecule variant or its corresponding GH polypeptide/protein variant, as afore described, for the diagnosis of growth hormone dysfunction or the development of suitable GH therapeutics.

According to a yet further aspect of the invention there is provided an antibody specific for the isolated growth hormone polypeptide or protein variant of the invention.

According to a further aspect of the invention there is provided a test kit suitable for carrying out the aforementioned second screening method of the invention which kit comprises:

(a) at least one antibody specific for one of the following substitutions in the growth hormone polypeptide/protein: Arg16His;

Phe176Cys; or Cys189Arg; and

(b) at least one antibody specific for the wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally, (c) one or more reagents suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

It will be apparent to those skilled in the art that a knowledge of the GH variants responsible for GH dysfunction is useful in the development of new therapeutics and therefore a method for the production of the novel GH variants described herein provides a suitable source of research material for use in identifying treatments for growth hormone dysfunction.

According to a further aspect of the invention there is therefore provided a vector encoding a variant of the nucleic acid molecule, GH1, according to the present invention which is for use in the production of a novel GH variant described herein which, in turn, is used, particularly, but not exclusively, as a research tool.

According a yet further aspect of the invention there is provided a vector encoding an oligonucleotide specific for at least one variant of the invention.

According to a further aspect of the invention there is provided a host cell transformed or transfected with the aforementioned vector.

According to a further aspect of the invention there is therefore provided a method for the production of a novel GH variant according to the invention comprising:

(i) culturing a host cell transformed or transfected with a vector encoding a GH1 variant according to the present invention; and

(ii) recovering from the culture the GH variant produced by said host cell.

According to a further aspect of the invention there is provided a polypeptide or protein extracted from culture medium and encoding a protein variant according to the invention.

According to a yet further aspect of the invention there is provided a screening method for screening an individual for the susceptibility to or existence of hypertension, which method comprises the steps of: (a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for the following mutation: T>C+1519; and (d) where said mutation exists concluding that said individual is likely to suffer from or is suffering from hypertension.

According to a further aspect of the invention there is provided a test kit suitable for screening an individual for the susceptibility to or existence of hypertension which kit comprises:

(a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH1 gene including the following mutation:

T>C+1519; (b) at least one oligonucleotide having a nucleic acid sequence complementary to the wild-type of the gene in the region specified in part (a); and, optionally,

(c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene.

According to a further aspect of the invention there is provided a screening method for screening an individual for the susceptibility to or existence of hypertension, which method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein;

(b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below;

(c) examining said sequence for the following substitution: Cys189Arg; and

(d) where said substitution exists concluding that the individual is likely to suffer from or is suffering from hypertension.

According to a further aspect of the invention there is provided a test kit suitable for screening an individual for the susceptibility to or existence of hypertension which test kit comprises:

(a) at least one antibody specific for the following substitution in the growth hormone polypeptide/protein:

Cys189Arg; and

(b) at least one antibody specific for wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally, one or more reagent suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

According to a further aspect of the invention there is provided the use of the nucleic acid mutation T>C+1519, or the corresponding protein substitution

Cys189Arg, for use in screening for, diagnosing or treating, hypertension.

According to a further aspect of the invention there is provided a screening method for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for the following mutation: T>G+1481 ; and

(d) where said mutation exists concluding that the individual has suffered a stroke or is likely to suffer a stroke.

According to a further aspect of the invention there is provided a test kit suitable for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which kit comprises:

(a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH1 gene including the following mutation:

T>G+1481 ;

(b) at least one oligonucleotide having a nucleic acid sequence complementary to the wild-type of the gene in the region specified in part (a); and, optionally, (c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene.

According to a further aspect of the invention there is provided a screening method for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein;

(b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below;

(c) examining said sequence for the following substitution: Phe176Cys; and

(d) where said substitution exists concluding that the individual has suffered a stroke or is likely to suffer a stroke.

According to a further aspect of the invention there is provided a test kit suitable for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which kit comprises:

(a) at least one antibody specific for the following substitution in the growth hormone polypeptide/protein:

Peh176Cys; and

(b) at least one antibody specific for the wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally,

(c) one or more reagents suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

According to a further aspect of the invention there is provided the use of T>G+1481 mutation or the corresponding protein variant Phe176Cys in the screening for, diagnosis of or treatment of, stroke.

Embodiments of the invention that will now be illustrated 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.Θ+/-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 a!., 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 antihypertensive 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 (PWV) 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.

Example 1 - 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 GH 1 promoter haplotypes. Polymerase chain reaction (PCR) amplification

PCR amplification of a 3.2 kb GH1 gene-specific fragment was performed using oligonucleotide primers GH1F (5' GGGAGCCCCAGCAATGC 3'; -615 to -599) and GH1 R (5' TGTAGGAAGTCTGGGGTGC 3'; 2598 to 2616) [numbering relative to the transcriptional initiation site at +1]. Briefly, reaction conditions for fragment generation consisted of amplifying 200ng lymphocyte DNA using the

Expand™ high fidelity system (Roche) using a hot start of 98⁰C 2 min, followed

by 95°C 3 min, 30 cycles 95⁰C 30 s, 64°C 30 s, 68°C 1 min. For the last 20

cycles, the elongation step at 68°C was increased by 5 s per cycle. This was

followed by further incubation at 68⁰C for 7 min.

Cloning and sequencing

GH1 -specific (3.2 kb) PCR fragments were directly sequenced with BidDye v2.0

(Applied Biosystems, Warrington, UK) according to the manufacturers recommendations and analysed on an ABI 3100 DNA sequencer. Primers used for sequencing were GH1 BF (5¹ GTGGTCAGTGTTGGAACTGC 3'; -556 to - 537), GH1 SEQ1 (5¹ CCACTCAGGGTCCTGTG 3'; +27 to +43), GH1SEQ2 (5^J GGAGGAGACTAAGGAGCTC 3'; +556 to +584), and GH1SEQ3 (5' TTAGAGAAACACTGCTGCCC 3'; +1137 to +1156) (Numbering relative to the transcriptional initiation site at +1 ; GenBank Accession No. J03071 ).

In vitro expression ofmissense GH variants

Wild-type GH1 cDNA was cloned into an insect expression vector and modified by site-directed mutagenesis to generate the novel missense GH variants as previously described (Millar et al., 2003). These vectors were then transfected into High Five insect cells (Invitrogen). Human GH was quantified in the culture supematants by ELISA (DRG Instruments GmbH, Marburg, Germany). The cross-reactivity in the ELISA of the GH variant and insect cell-expressed wild- type GH was confirmed by dilutional analysis to be equal to that of the assay reference preparation (calibrated against the MRC 1st IRP 80/505 reference preparation).

Luciferase reporter gene assay of STAT 5 activation HK293 cells, previously transfected with the full-length human GHR gene and selected on the basis of elevated GH receptor expression (HK293hi), were used to assay STAT 5 activation (Ross et al., 1997; von Laue et al., 2000). HK293N cells were transfected with a STAT 5-responsive luciferase reporter gene construct and treated with GH (wild-type and variant) for 6 hours. Luciferase expression was measured as previously described (Lewis et al., 2004).

GH secretion studies in mammalian cells

Rat pituitary (GC) cells were transfected with a pGEM-T plasmid containing a 3.2 kb fragment spanning the entire wild-type GH1 gene or the equivalent construct for the missense variants. These were assayed as previously described (Lewis et al., 2004). Human GH in the GC cell culture supernatant was assayed using an ELISA specific for human GH (DRG, Marburg). Characterization of novel GH variants detected

During the course of our studies, three novel GH variants were identified: Arg16His (control individual 59, hypertension study), Phe176Cys (stroke patient 76) and Cys189Arg (hypertension patient 73); intriguingly, the sites of all three mutated residues are known to be involved in the binding of GH to its receptor

(GHR).

Arg16 (helix 1 ) is solvent-accessible and forms part of binding site 2 (De Vos et al., 1992), interacting with Tyr169 of the GHR. In vitro substitution of alanine for arginine at this position has a significant effect on the binding of the extracellular domain of the GHR to GH site 2, resulting in a 13-fold increase in K_d compared to that of wild-type GH (Walsh et al., 2003). Substitution by His could adversely affect site 2-binding although, owing to the structural similarity of Arg and His, it is likely that this substitution will have a relatively small effect on the interaction of GH with its receptor. This notwithstanding, Arg16 is evolutionarily conserved in GH from all vertebrates examined and a naturally occurring Arg16Cys variant manifesting reduced GH secretion has been previously reported in a control individual (Millar et al., 2003). The His16 variant required a higher concentration of the variant than wild-type GH (4.7±0.8nM vs. 2.3±0.29nM, P<0.01 , n=3) to produce half maximal effect (EC50) in a STAT5-responsive reporter gene assay, resulting in reduced biological activity (39±11.5% wild-type at EC50 of wild-type GH, P<0.01 , n=3). Secretion was however found to be normal (127±22% wild- type). Phe176 is located in helix 4 of GH and forms part of binding site 1. Studies using alanine-scanning mutagenesis have identified Phe176 as one of eight key residues that account for 85% of the binding energy of the GH binding site 1- GHR interaction (Wells, 1996). Substitution of alanine for phenylalanine at this position leads to a 22-fold increase in the rate of dissociation of GH from its receptor (Cunningham and Wells, 1993) resulting in an overall 16-fold increase in K_d (Cunningham and Wells, 1989). It would therefore appear likely that the substitution of phenylalanine by cysteine at position 176 will perturb the binding of the GH variant to the GHR. Additionally, the presence of a cysteine residue at this position may allow the formation of inappropriate disulphide bonds that will alter GH structure and hence further disrupt the variant GH-GHR interaction. The aromatic and hydrophobic Phe176 residue is actually human-specific, the analogous residue being Tyr in all other vertebrates examined (in evolutionary terms, however, Phe176 in humans constitutes a fairly conservative substitution). The Cys176 variant was found to have a higher EC5₀ than wild- type GH (4.9+0.56nM vs. 2.3±0.29nM, PO.01, n=3) resulting in reduced biological activity (43±7.1% wild-type at EC₅₀ of wild-type GH, P=0.01 ). Secretion of Cys176 was however found to be normal (106±29% wild-type).

Cys189 forms a disulphide bond with Cys182 to create a small C-terminal loop.

By means of site-directed mutagenesis, the replacement of either of these Cys residues by Ser has been shown to abrogate small loop formation, and whilst this small loop is non-essential for GH biological activity (Graf et al., 1975; 1976; Chen et al., 1992), the Ser variants display reduced affinity for the GHR (Chen et al., 1992). Since Cys189 forms part of the binding site 1 of GH and interacts closely with the GHR (De Vos et al., 1992), it may be that the substitution of arginine for a cysteine residue at this position alters the GH-GHR interaction. In support of this view, Cys189 is present at the analogous position in all other vertebrates examined. The Arg189 variant is however indistinguishable from wild-type in terms either of its secretion (135±17% wild-type) or of its ability to activate STAT5 (EC₅O = 2.6±0.37nM vs. 2.3±0.29nM for wild-type), consistent with the early in vitro findings cited above.

During the course of our study, three novel heterozygous GH coding sequence variants were identified. A knowledge of these three variants is important in the understanding of growth hormone function and the repercussive effects of this important endocrine molecule on, historically, seemingly unrelated conditions such as hypertension and stroke.

References

Britton J, Pavord I₁ Richards K, Wisniewski A, Knox A, Lewis S, Tattersfield A, Weiss S. (1994) Dietary magnesium, lung function, wheezing, and airway hyperreactivity in a random adult population sample. Lancet 344: 357-362.

Chen XZ₁ Shafer AW, Yun JS, Li YS₁ Wagner TE₁ Kopchick JJ. (1992) Conversion of bovine growth hormone cysteine residues to serine affects secretion by cultured cells and growth rates in transgenic mice. MoI. Endocrinol. 6: 598-606.

Cunningham BC, Wells JA. (1989) High-resolution epitope mapping of hGH- receptor interactions by alanine-scanning mutagenesis. Science 244: 1081- 1085. Cunningham BC, Wells JA. (1993) Comparison of a structural and a functional epitope. J. MoI. Biol. 234: 554-563.

De Vos AM, Ultsch M, Kossiakoff AA. (1992) Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255: 306-312.

Graf L, Li CH, Bewley TA. (1975) Selective reduction and alkylation of the COOH-terminal disulfide bridge in bovine growth hormone. Int. J. Pept. Protein Res. 7: 467-473.

Graf L, Borvendeg J, Barat E, Hermann I₁ Patthy A. (1976) Reactivity and biological importance of the disulfide bonds in human growth hormone. FEBS Letts. 66: 233-237. Horan M, Millar DS, Hedderich J, Lewis G, Newsway V₁ Mo N, Fryklund L,

Procter AM, Krawczak M, Cooper DN. (2003) Human growth hormone 1 (GH1) gene expression is influenced in a complex haplotype-dependent fashion by polymorphic variation in both the proximal promoter and the locus control region. Hum. Mutation 21 : 408-423.

Lewis MD, Horan M, Millar DS, Newsway V, Easter TE, Fryklund L, Gregory JW, Norin M₁ del VaIIe C-J, Lόpez-Siguero JP, Lόpez-Canti LP₁ Diaz-Torrado N₁ Espino R₁ Ulied A, Scanlon MF, Procter AM₁ Cooper DN. (2004) A novel dysfunctional GH variant (lle179Met) exhibits a decreased ability to activate the extracellular signal-regulated kinase (ERK) pathway. J. Clin. Endocrinol. Metab.

89: 1068-1075.

McEniery CM, Yasmin, Hall IR, Qasem A₁ Wilkinson IB₁ Cockcroft JR (2005) Normal vascular aging: differential effects on wave reflection and aortic pulse wave velocity. The Anglo-Cardiff Collaborative Trial (ACCT). J. Am. Coll.

Cardiol. In press. Millar DS, Lewis MD, Horan M, Newsway V, Easter TE, Gregory JW₁ Fryklund L, Norin M, Crowne EC, Davies SJ, Edwards P, Kirk J₁ Waldron K, Smith PJ, Phillips JA 3rd, Scanlon MF, Krawczak M, Cooper DN₁ Procter AM. (2003) Novel mutations of the growth hormone 1 (GH 1) gene disclosed by modulations of the clinical selection criteria for individuals with short stature. Hum. Mutation 21 : 424-440.

O'Brien E, Mee F, Atkins N, Thomas M. (1996) Evaluation of three devices for self measurement of blood pressure according to the revised British

Hypertension Society Protocol: the Omron HEM-705CP, Philips HP5332, and Nissei DS-175. Blood Press. Monitoring 1 : 55-61.

Pauca AL, O'Rourke MF, Kon ND. (2001) Prospective evaluation of a method for estimating ascending aortic pressure from the radial artery pressure waveform.

Hypertension 38: 932-937.

Ross RJ, Esposito N, Shen XY, von Laue S, Chew SL, Dobson PR₁ Postel- Vinay MC₁ Finidori J. (1997) A short isoform of the human growth hormone receptor functions as a dominant negative inhibitor of the full-length receptor and generates large amounts of binding protein. MoI. Endocrinol. 11 : 265-273. von Laue S₁ Finidori J, Maamra M₁ Shen X-Y, Justice S, Dobson PRM, Ross RJM. (2000) Stimulation of endogenous GH and interleukin-6 receptors selectively activates different Jaks and Stats, with a Stat5-specific synergistic effect of dexamethasone. J. Endocrinol. 165: 301-311.

Walsh STR, Jevitts LM₁ Sylvester JE, Kossiakoff AA. (2003) Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization. Protein Sci. 12: 1960-1970.

Wells JA. (1996) Binding in the growth hormone receptor complex. Proc. Natl. Acad. Sci. USA 93: 1-6. Wilkinson IB₁ Fuchs SA₁ Jansen IM, Spratt JC, Murray GD, Cockcroft JR, Webb

DJ. (1998) The reproducibility of pulse wave velocity and augmentation index measured by pulse wave analysis. J. Hypertens. 16: 2079-2084.

Claims

1. An isolated variant of the growth hormone nucleic acid molecule, GH1, comprising one or more of the following substitutions: G>A+447 (Arg16His), T>G+1481 (Phe176Cys) and T>C+1519 (Cys189Arg).

2. An isolated variant according to claim 1 wherein said nucleic acid molecule is either DNA or RNA.

3. An isolated polypeptide or protein which is a variant of the growth hormone protein, GH, and which comprises any one or more of the following substitutions:

Arg16His

Phe176Cys Cys189Arg.

4. A screening method for screening an individual suspected of having dysfunctional GH which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for any one or more of the following substitutions: G>A+447, T>G+1481 and T>C+1519; and (d) where one or more of said substitutions exist concluding that the individual suffers from or is likely to suffer from GH dysfunction.

5. A screening method according to claim 4 wherein said nucleic acid molecule comprises either DNA or RNA.

6. A screening method for screening an individual suspected of having GH dysfunction, which method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein;

(b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below;

(c) examining said sequence for one or more of the following substitutions: Arg16His; Phe176Cys; or

Cys189Arg; and

(d) where one or more of said substitutions exist concluding that the individual suffers from or is likely to suffer from GH dysfunction.

7. A test kit suitable for carrying out the screening method according to claim 4, which kit comprises:

(a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH1 gene including one of the following substitutions: G>A+447, T>G+1481 and T>C+1519; (b) at least one oligonucleotide having a nucleic acid sequence complementary to the wild-type of the gene in one of the regions specified in part (a); and, optionally,

(c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene.

8. A test kit according to claim 7 wherein said oligonucleotide comprises at least one PCR primer described herein.

9. A test kit suitable for carrying out the screening method according to claim 6, which kit comprises:

(a) at least one antibody specific for one of the following substitutions in the growth hormone polypeptide/protein:

Arg16His; Phe176Cys; or

Cys189Arg; and

(b) at least one antibody specific for the wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally,

(c) one or more reagents suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

10. Use of a GH1 nucleic acid molecule variant according to claim 2 or its corresponding GH polypeptide/protein variant according to claim 3, for the diagnosis of growth hormone dysfunction or the development of suitable GH therapeutics.

11. A vector encoding a variant of the nucleic acid molecule, GH1, according to claim 1.

12. Use of a vector according to claim 11 for the production of a novel GH variant according to claim 3.

13. A vector encoding an oligonucleotide specific for at least one variant according to claim 1.

14. A host cell transformed or transfected with the vector according to claims 11 or 13.

15. A method for the production of a novel GH variant according to claim 1 comprising:

(i) culturing a host cell transformed or transfected with a vector encoding a nucleic acid molecule according to claim 1 ; and

(ii) recovering from the culture the GH variant produced by said host cell.

16. A polypeptide or protein extracted from culture medium and encoding a protein variant according to claim 3.

17. A screening method for screening an individual for the susceptibility to or existence of hypertension, which method comprises the steps of:

(a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for the following mutation: T>C+1519; and

(d) where said mutation exists concluding that said individual is likely to suffer from or is suffering from hypertension.

18. A test kit suitable for screening an individual for the susceptibility to or existence of hypertension which kit comprises:

(a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH1 gene including the following mutation:

T>C+1519; (b) at least one oligonucleotide having a nucleic acid sequence complementary to the wild-type of the gene in the region specified in part (a); and, optionally,

(c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene.

19. A screening method for screening an individual for the susceptibility to or existence of hypertension, which method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein; (b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below;

(c) examining said sequence for the following substitution: Cys189Arg; and

(d) where said substitution exists concluding that the individual is likely to suffer from or is suffering from hypertension.

20. A test kit suitable for screening an individual for the susceptibility to or existence of hypertension which test kit comprises:

(a) at least one antibody specific for the following substitution in the growth hormone polypeptide/protein:

Cys189Arg; and

(b) at least one antibody specific for wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally, one or more reagent suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

21. Use of the nucleic acid mutation T>C+1519, or the corresponding protein substitution Cys189Arg, for use in screening for, diagnosing or treating, hypertension.

22. A screening method for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising GH1 nucleic acid molecule;

(b) sequencing at least a part of said molecule in order to undertake (c) below;

(c) examining said sequence for the following mutation: T>G+1481 ; and (d) where said mutation exists concluding that the individual has suffered a stroke or is likely to suffer a stroke.

23. A test kit suitable for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which kit comprises: (a) at least one oligonucleotide having a nucleic acid sequence complementary to a region of the GH1 gene including the following mutation: T>G+1481 ;

(b) at least one oligonucleotide having nucleic acid sequence complementary to the wild-type of the gene in the region specified in part (a); and, optionally, (c) one or more reagents suitable for carrying out amplification and/or sequencing of said gene.

24. A screening method for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which screening method comprises the steps of:

(a) obtaining from said individual a test sample comprising growth hormone polypeptide/protein;

(b) sequencing at least a part of said polypeptide/protein in order to undertake (c) below; (c) examining said sequence for the following substitution: Phe176Cys; and

(d) where said substitution exists concluding that the individual has suffered a stroke or is likely to suffer a stroke.

25. A test kit suitable for screening an individual suspected of having suffered a stroke or suspected of being susceptible to stroke which kit comprises: (a) at least one antibody specific for the following substitution in the growth hormone polypeptide/protein: Peh176Cys; and (b) at least one antibody specific for the wild-type of the growth hormone polypeptide/protein in the region referred to in (a); and, optionally, (c) one or more reagents suitable for providing conditions that enable the binding of the above antibody or antibodies to the said polypeptide/protein.

26. Use of T>G+1481 mutation, or the corresponding protein variant

Phe176Cys, in the screening for, diagnosis of or treatment of, stroke.

27. A primer for use in the method according to claim 4, 17, 22 or the test kit according to claim 7, 18, 23 wherein said primer is selected from one of the following: GH1 BF (5' GTGGTCAGTGTTGGAACTGC 3'; -556 to -537),

GH1 SEQ1 (5¹ CCACTCAGGGTCCTGTG 3'; +27 to +43), GH1 SEQ2 (5^J GGAGGAGACTAAGGAGCTC 3'; +556 to +584), and GH1 SEQ3 (5¹ TTAGAGAAACACTGCTGCCC 3'; +1137 to +1156).