WO2002090585A2 - Genetic polymorphism pattern of tgfb1 as marker for susceptability to renal disease - Google Patents

Abstract

Methods and kits are disclosed for detecting an individual's susceptibility to renal disease. Methods comprise obtaining a biological sample from an individual and determining the presence or absence of a genetic polymorphism pattern in TGFB1, which polymorphism pattern is associated with progression of renal failure.

Description

GENETIC MARKER

The present invention relates to a method of detecting the susceptibility of an individual to the progression of renal disease. More specifically, the invention relates to polymorphisms in a cytokine gene and the genetic association of these polymorphisms with the progression of chronic renal failure.

Chronic renal failure (CRF) is defined as an irreversible, long-standing loss of renal function. End stage renal failure (ESRF) is an advanced form of CRF and refers to advanced renal insufficiency when renal function is approximately 10% of normal prior to the initiation of either dialysis or renal transplantation (Εl-Nahas and inearls, 1991 ) . A U.K survey of hospital biochemistry records revealed that the prevalence of CRF is 2058 adults per million population (p.m.p). This was determined by measuring the concentration of the protein creatinine in the blood plasma. A concentration of creatinine greater than >150 μmol/1 in the plasma is indicative of CRF. The data also revealed the incidence of ESRF to be 78 p.m.p/year (Geerling, et al . , 1994).

Studies have shown that the incidence of renal failure from various renal diseases increases with age (Feest, et al . , 1990, McGowen, 1990 and Junger, et al . , 1996) and that vascular diseases and diabetes mellitus are the most common causes of ESRD in the elderly (Malberti, et al . , 1997) . CRF can also arise as a complication of systemic lupus erythematosus, obstruction to the urinary tract or inflammation of the tubular system (pyelonephritis) . Furthermore, the patient's sex and race are important in progression of renal failure since, whatever the cause of renal failure, ESRD is more common in males than in females, and African-Americans show a four-fold higher incidence of ESDR levels than that of Caucasians. Furthermore, ESRD is higher in Native Americans and Asian/Pacific Islanders than Caucasians. The greater risk of ESRD in African-Americans may be due to a genetic predisposition (Freedman et al . , 1997) .

Systemic hypertension has been shown to be one of the major factors contributing to the deterioration of renal function and elevated blood pressure can be a cause or a consequence of renal injury. The incidence of systemic hypertension in patients with serious renal disease is 90% (Lazarus, et al . , 191 A) . Furthermore, the incidence of renal failure in patients with non-accelerated essential (no apparent cause) hypertension is about 10% (Michael, et al . , 1980; Meyer et al . , 1985). Therefore, the measurement of the systolic and diastolic blood pressure may be used as an indicator of renal disease.

Pharmacological interventions that reduce the concentration of protein in the urine (proteinurea) have been shown to be associated with slowing down the progression of CRF (Bjorck, et al . , 1986 and Apperloo, et al . , 1994). Furthermore, the presence of proteinuria has been shown to predict renal failure in patients with chronic pyelonephritis (Kincaid-Smith & Becker, 1978). Therefore, the detection of proteinuria is also an important prognostic indicator of renal disease.

One of the primary components of inflammatory and other immune responses is cytokine production. Cytokines are soluble peptide/protein immunomodulators that act as messengers between cells and have been shown to play a role in the initiation of fibrosis of the kidney. Cytokines are produced by tissue cells and activated immune cells including thymus-derived lymphocytes (T- cells) , B lymphocytes and monocyte/macrophages . Medcraft, et al . (1993), showed that there was a relationship between genetic polymorphisms in different pro- and anti- inflammmatory cytokines and the severity of renal diseases. For example, a polymorphism in the TNFα gene has been found to be associated with immunoglobulin A (IgA) nephropathy, idiopathic membranous nephropathy and Type I (insulin-dependent) diabetes mellitus. Furthermore, it was demonstrated that the interleukin 1 receptor antagonist allele 2 (IL-1RN*2) could be used as a marker for severity or risk of complications of inflammatory processes, including diabetic nephropathy (Blakemore, et al . , 1996). In addition, Freedman, et al . , (1997) showed that there was an association between IL-1RN*2 and end stage renal disease.

Another cytokine that has been shown to play a role in the initiation of fibrosis of the kidney is transforming growth factor beta (TGFβ) which has three subclasses βl, β2 and β3. These stimulate the production of the fibronectin, collagen, and proteoglycan in fibroblasts during the normal repair process that follows tissue injury (Border, et al . , 1990). TGFβ plays an important role in both glomerular sclerosis and tubulointerstitial scarring by inducing monocytic and lymphocytic glomerular infiltration, mesangial proliferation and sclerosis (Johnson, et al . , 1991 and Savage, 1994). Genetic testing (also referred to as genetic screening, genotyping or molecular diagnostics) may be defined as the testing of the nucleic acid of an individual or patient in an analytical capacity to determine whether the patient carries polymorphic alleles or mutations causing, or associated with, a disease state or linked to a mutation or allele causing, or associated with, the disease. The number of diseases in which genetic testing and other biological methods are informative is continually increasing. The early detection of a pre-disposition to a genetic disease presents the best opportunity for medical intervention. In addition, markers associated with an increased risk of severe clinical course of a particular disease may be screened. Early genetic diagnosis and screening for markers associated with disease severity, complications or rate of progression may improve the prognosis for a patient through supervision and early intervention before the clinically detectable disorder occurs. In cases where patients with similar symptoms are treated with variable success, sophisticated genetic testing can differentiate individual patients with subtle or undetectable differences and can lead to more suitable individual treatments. It is even possible that early intervention may eventually involve methods such as gene therapy.

Unfortunately, to date, it has not been possible to utilise genetic markers in assessing the risk to a patient of the progression of renal disease. Therefore, it is one of the aims of embodiments of the present invention to address the problems outlined above and to provide a method by which physicians can assess the risk to a patient of the progression of chronic renal failure and which could thereby act as an early indication for increased monitoring and proactive clinical management. It is a further aim of the invention to provide a kit by which physicians can detect the susceptibility of a patient to the progression of chronic renal failure. The ability to identify high-risk individuals by such detection systems would allow physicians to focus preventative measures on those individuals who may gain the greatest benefit, i.e. those patients with higher risk of progression of chronic renal failure, and would provide strong incentives for those at higher risk to comply with such approaches so as to slow down or prevent progression of the disease to end stage renal disease.

For convenience, the meaning of certain terms and phrases employed in the specification, examples and claims are provided below. In addition, these terms and phrases should be understood in relation to the specification as a whole.

The term "polymorphism" refers to the co-existence, within a population, of more than one form of a gene or portion thereof (e.g. allelic variant), at a frequency too high to be explained by recurrent mutation alone. A portion of a gene of which there are at least two different forms, i.e. two different nucleotide sequences, is referred to as a "polymorphic region of a gene". A specific genetic sequence at a polymorphic region of a gene is an allele. A polymorphic region can be a single nucleotide, the identity of which differs in different alleles. A polymorphic region can also be several nucleotides long. Techniques for determining the presence of particular alleles would be those known to persons skilled in the art and include, but are not limited to, nucleic acid techniques based on size or sequence, such as restriction fragment length polymorphism (RFLP) , nucleic acid sequencing, or nucleic acid hybridization. The nucleic acid tested may be RNA or DNA. These techniques may also comprise the step of amplifying the nucleic acid before analysis. Amplification techniques are known to those of skill in the art and include, but are not limited to, cloning, polymerase chain reaction (PCR) , polymerase chain reaction of specific alleles (PASA) , polymerase chain ligation, nested polymerase chain reaction, and the like. Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele- specific oligonucleotide (ASO) hybridization, allele specific 5' exonuclease detection, sequencing, hybridization and the like. Polymorphic variations leading to altered protein sequences or structures may also be detected by analysis of the protein itself.

The term "allele" refers to the different sequence variants found at different polymorphic sites in DNA obtained from a subject. For example, each polymorphic region of the TGFBl gene has at least two different alleles, ie . first and second alleles at the first and second polymorphic regions. The sequence variants may be single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats.

The term "marker" refers to a sequence in the genome that is known to vary among individuals, ie. a polymorphic region which is used as a tool in genetic analysis. For example, the TGFBl gene contains several potential markers. The markers discussed hereinafter are TGFBl Arg25Pro and TGFBl promoter T-509C. Either marker, as described herein, may be used for identification of individuals at higher risk of the progression of renal disease from CRF to ESRD.

The two polymorphisms/alleles/markers of the TGFBl gene herein referred to may be illustrated as follows :-

1) Codon 25 of TGFBl: Arg25Pro

The alleles of a bi-allelic polymorphism of a single base variation (G/C) in the 2^nd position of codon 25 are identified as : -

G-allele (first allele) : CGG Arginine

C-allele (second allele) : CCG Proline

In the G-allele (also hereinafter referred to as the first allele of the Arg25Pro polymorphism) , the nucleotide in the 2^nd position of the 25th codon of exon 1 of TGFBl is a guanine and the codon encodes the amino acid arginine. For reference, SEQ ID NO's:6 & 8 show the nucleic acid and protein sequences of the TGFBl gene with the G-allele, respectively. In the C-allele (also hereinafter referred to as the second allele of the Arg25Pro polymorphism) , the nucleotide in the 2^nd position of the 25th codon of exon 1 of TGFBl is a cytosine and the codon encodes the amino acid proline. For reference, SEQ ID NO's:7 & 9 show the nucleic acid and protein sequences of the TGFBl gene with the C-allele, respectively. 2) TGFBl promoter at Thymine (T) -509 Cytosine (C)

The C-allele (also hereinafter referred to as the first allele of the T(-509)C polymorphism) comprises a cytosine base at the -509 nucleotide position.

The T-allele (also hereinafter referred to as the second allele of the T(-509)C polymorphism) comprises a thymine base at the -509 nucleotide position.

The terms "genotype", "allelic pattern" or "polymorphism pattern" refer to the identity of an allele or alleles at one or more polymorphic sites. A genotype, allelic pattern or polymorphism pattern may consist of either a homozygous or heterozygous state at one or more polymorphic sites. For example, TGFBl Arg25Pro (G,G) is a genotype, allelic pattern or polymorphism pattern in which there are two copies of the G-allele at the Arg25Pro marker of TGFBl corresponding to the homozygous TGFBl G-allele state. Alternatively, an individual may be homozygous for the C- allele. Furthermore, TGFBl Arg25Pro (G,C) is a genotype in which there is one copy of the G-allele and one copy of the C-allele corresponding to the heterozygous state.

The term "haplotype" refers to a set of alleles that are inherited together as a group (they are in linkage) . As used herein, haplotype is defined to include those haplotypes that occur at statistically significant levels (Pcorr≤O .05) . As used herein, the phrase a "TGFBl haplotype" refers to a haplotype at the TGFBl locus.

"Linkage disequilibrium" refers to the co-inheritance of two or more alleles at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given control population. The expected frequency of occurrence of two alleles that are inherited independently is the frequency of the first allele in that population multiplied by the frequency of the second allele in that population. Alleles that co-occur at expected frequencies are said to be in "linkage equilibrium". The cause of linkage disequilibrium is often unclear. It can be due to selection for certain allele combinations or to recent admixture of genetically heterogeneous populations. In addition, in the case of markers that are very tightly linked to a disease gene, an association of an allele (or group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the specific chromosomal region. When referring to allelic patterns or polymorphism patterns that comprise more than one allele, a first allelic pattern is in linkage disequilibrium with a second allelic pattern if at least one of the alleles that comprise the first allelic pattern are in linkage disequilibrium with at least one of the alleles of the second allelic pattern. Such an example of linkage disequilibrium would be that which occurs between the alleles at the Arg25Pro and T(-509)C polymorphic sites, for example, the C-allele of Arg25Pro and the T-allele of T(-509)C.

"Allele detection" refers to any means known to those skilled in the art of detecting or differentiating between alleles, e.g. detecting whether the allele at any given position of the TGFBl gene is allele 1 or 2, for example, the G-allele or the C-allele of Arg25Pro. We describe herein at least two means of determining which allele is present in a population. Firstly, PCR amplification of a specific polymorphic region followed by digestion of the PCR product and size fractionation. Secondly, PCR amplification of a specific polymorphic region using Amplification Refractory Mutation System (ARMS) . However, numerous techniques for detecting a specific allele are known and need not be described herein.

The term "detecting alleles" refers to the process of genotyping, genetic testing, genetic screening, determining or identifying an allele or polymorphism. The allele actually detected might be a disease-causing mutation or a disease-associated polymorphic allele (eg C- allele of Arg25Pro, or T-allele of T-509C) , or a mutation that is in linkage disequilibrium with a disease-causing mutation. It will manifest in the genomic DNA of an individual, but may also be detectable from RNA or protein sequences transcribed or translated from the region.

As used herein, the term "nucleic acid" refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA) , and, where appropriate, ribonucleic acid (RNA) . The term should also be understood to include, as equivalents, analogues of either RNA or DNA made from nucleotide analogues and as applicable to the embodiment being described, single- stranded (sense or antisense) and double-stranded polynucleotides .

The term "polymerase chain reaction" or "PCR" refers to a method of amplifying DNA, preferably, small amounts of DNA for ease of analysis. Many variations of the basic amplification protocol are well known to those of skill in the art. PCR-based detection means include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analysed simultaneously. Alternatively, it is possible to amplify different markers with primers that are differentially labelled and thus can each be differentially detected. Of course, hybridization-based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known in the art to allow multiplex analysis of a plurality of markers.

The term "susceptibility", "propensity" or "predisposition" to disease or any similar phrase, means that certain alleles are hereby discovered to be associated with, or predictive of, progression of renal disease from CRF to ESRD. The alleles are thus over- represented in frequency or carriage rate in individuals who show a progression from CRF to ESRD as compared to individuals whose condition does not progress from CRF to ESRD.

The patient's "susceptibility to progression of renal failure" refers to a statistically higher frequency or rate of progression of renal failure in an individual carrying a particular polymorphic allele, or genotype (allelic or polymorphism pattern) in comparison to the frequency or rate of progression in a member of a population that does not carry the particular polymorphic allele, or genotype (allelic or polymorphism pattern) . Preferably, "susceptibility" refers to a progression of renal failure from a state of chronic renal failure (CRF) to the development of End Stage Renal Disease (ESRD) .

According to a first aspect of the present invention, there is provided a method for determining an individual's susceptibility to renal failure, said method comprising: - (i) obtaining a sample from an individual; and (ii) detecting the presence of a genetic polymorphism pattern in TGFBl gene in said sample which polymorphism pattern is associated with renal failure.

Preferably, said susceptibility to renal failure means the individual is susceptible to progression of renal failure.

The TGFBl gene is located on chromosome 19ql3.1-13.3 (Fujii et al . , 1986). The coding DNA sequence of TGFBl is known and is readily accessible at www.ncbi.nlm.nih.gov (Accession Number: X02812) and has been published by Derynck R et al . (1985). This sequence is incorporated herein by reference, and shown in the sequence listing.

Preferably, the polymorphism pattern comprises at least one polymorphism or polymorphic region of the TGFBl gene. Preferably, the polymorphism pattern comprises more than one polymorphic region of the TGFBl gene.

Preferably, the method of the invention comprises determining whether the individual is homozygous or heterozygous for alleles at polymorphic regions of the TGFBl gene or other linked regions thereof. Preferably, the method is conveniently used to screen for an individual at risk to renal failure correlated with the polymorphism pattern of said TGFBl gene.

Preferably, said polymorphism is selected from any of the following polymorphisms:- at position -988 (C/A) , -800 (G/A) , -509 (C/T) upstream of the coding region of the TGFBl gene, codon 10 (LeulOPro) of exon 1 of the TGFBl gene, codon 25 (Arg25Pro) of exon 1 of the TGFBl gene, codon 263 of exon 5 of the TGFBl gene, and a (C) insertion site at the 3' at position 72 of the non-translated region of said TGFBl gene. The polymorphism pattern may comprise a further polymorphism which is in linkage disequilibium with the polymorphism which is known to be associated with renal failure.

In a preferred embodiment, any two of said polymorphic regions correlate with the progression of renal disease. Preferably, at least two of said polymorphisms are in linkage disequilibrium with each other. Preferably, the first polymorphism comprises a first allele and a second allele. Preferably, the second polymorphism comprises a first and a second allele. Preferably, the second alleles of the first and the second polymorphic regions correlate with the progression of renal failure.

Preferably, and advantageously, diagnosis of the progression of renal disease may be carried out by detection of either or both of these two polymorphic regions, more preferably, either or both of the second alleles of these two polymorphic regions. Preferably, diagnosis of the progression of renal disease may be carried out by detecting which alleles of the two polymorphic regions are present. Advantageously, screening for the presence of either or both of first and second alleles of the polymorphic regions allows for the identification of individuals likely to have a genetic susceptibility to the progression of renal failure.

Preferably, the polymorphism pattern comprises a polymorphism in exon 1 of the TGFBl gene, preferably, an Arg25Pro polymorphism in exon 1 of the TGFBl gene. The first allele of the Arg25Pro polymorphism is a guanine base, and the second allele of the Arg25Pro polymorphism is a cytosine base. Preferably, the second allele of the Arg25Pro polymorphism correlates with progression of renal disease .

Alternatively, or additionally, the polymorphism pattern may comprise a polymorphism in the promoter region of the TGFBl gene. Preferably, the polymorphism in the promoter region is at the -509 nucleotide position and is, more preferably, a thymine/cytosine (T/C) polymorphism. The first allele of -509 nucleotide position polymorphism is a cytosine base, and the second allele of -509 nucleotide position is a thymine base. Preferably, the second allele of the -509 nucleotide position polymorphism correlates with progression of renal disease.

Preferably, in carrying out the method of the invention, an individual's TGFBl gene genotype is determined by analysis of, preferably, a region of the TGFBl gene, rather than by analysis of the entire gene sequence.

Preferably, in an embodiment of the present invention, the susceptibility is assessed by determining whether an individual is homozygous or heterozygous for either or both of the second alleles of the first and second polymorphic regions of the TGFBl gene.

According to the invention, an individual who carries either or both of the second alleles is classified as being at a higher risk for progression of renal disease when compared against an individual who has the first allele for the polymorphic regions . In this specific embodiment, the presence of either or both of the second alleles correlates with a susceptibility to progression of renal failure.

Preferably, the sample comprises a biological sample which, preferably, comprises nucleic acid.

Preferably, the nucleic acid encodes at least the TGFBl gene and, more preferably, at least exon 1 of the TGFBl gene and, most preferably, the 25^th codon of exon 1 of the TGFBl gene. Alternatively, or additionally, the sample encodes at least the region upstream of the start codon of the TGFBl gene and, preferably, at least the promoter region of the TGFBl gene and, more preferably, at least the -509 nucleotide position of the TGFBl gene promoter region.

Preferably, the sample comprises genomic DNA and, more preferably, comprises at least the 25^th codon of exon 1 and the -509 nucleotide region of the promoter of the TGFBl gene.

Preferably, said detecting comprises amplification, more preferably, PCR amplification of the sample. Preferably, said detecting comprises ARMS (amplification refractory mutation system) PCR of the sample when detecting for the Arg25Pro polymorphism.

Preferably, said detecting comprises amplifying at least the region encoding exon 1 and, preferably, codon 25 of the TGFBl gene and identifying the allele encoded by said amplified DNA. Alternatively, or additionally, said detecting, preferably, comprises amplifying at least the promoter region of the TGFBl gene, preferably, the -509 nucleotide of the TGFBl gene and identifying the allele encoded by said amplified DNA.

Preferably, said detecting comprises use of at least one oligonucleotide operable to be used for amplification of genomic DNA encoding exon 1 of the TGFBl gene. Preferably, said detecting comprises use of at least one oligonucletide operable to be used for amplification of genomic DNA encoding the upstream region and, preferably, the promoter region of the TGFBl gene.

Preferably, the PCR amplification employs at least one primer comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

Preferably, the PCR amplification employs at least one primer comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO.2 and SEQ ID NO.3 when detecting for the Arg25Pro polymorphism, and preferably, at least one primer comprising a sequence selected from either SEQ ID NO.4 or SEQ ID NO.5 when detecting for the T(-509)C promoter polymorphism. Preferably, said detecting comprises subjecting the amplified DNA to size analysis, preferably, electrophoresis and, preferably, comparing the results to a positive control and, preferably, a negative control.

Said size analysis may be preceded by restriction enzyme digestion. Said detecting may comprise digesting the amplified DNA with a restriction enzyme, preferably, _3su36I, and then, preferably, subjecting the resultant digested DNA to electrophoresis and, preferably, comparing the results to a positive and, preferably, a negative control .

Preferably, and advantageously, the alleles of a bi- allelic polymorphism of a single base variation (T/C) at T(-509)C are identified by allele-specific cleavage using a restriction enzyme, preferably, _3su36l. After restriction enzyme digestion of the products of the PCR reaction, the DNA may be separated on a gel by electrophoresis. From this gel, the alleles of the polymorphism may be identified.

Alternatively, or additionally, the gel may undergo Southern blotting or other hybridization analyses comprising labeling, preferably, radio-labeling a probe.

Said detecting may comprise sequencing the DNA encoding the polymorphisms to determine the allele or alleles present.

According to a further aspect of the present invention there is provided a kit for identifying an individual ' s genetic polymorphism pattern in TGFBl gene which pattern is associated with renal disease, the kit comprising: -

(i) means for determining the presence of a genetic polymorphism pattern in TGFBl gene, wherein said polymorphism pattern is associated with progression of renal disease.

Preferably, said susceptibility to renal failure means the individual is susceptible to progression of renal failure.

Preferably, the kit further comprises DNA sample collecting means. Preferably, the means for determining the presence of the polymorphism pattern comprises analysis of said DNA sample, more preferably, genetic analysis .

Preferably, the kit further comprises means to compare the pattern to a control sample of known disease severity to determine an individual' s susceptibility to progression of renal disease.

The DNA sample collecting means may be suitable for isolating a DNA sample from the individual from which DNA may be used for subsequent analysis. The DNA sample may be obtained from a tissue sample, for example, blood, saliva, or urine etc.

In a preferred embodiment, the DNA is obtained from blood cells, preferably, obtained from a finger prick of the individual. Preferably, the DNA sample collecting means is operable to isolate blood from the individual. Preferably, the DNA is isolated from dried blood spots. Preferably, the DNA comprises target sequences which are, preferably, amplified using PCR. Preferably, said target sequences comprise the polymorphism pattern.

The polymorphism pattern may comprise at least one polymorphism and/or polymorphic region of the TGFBl gene. Preferably, the polymorphism pattern comprises a plurality of polymorphic regions of the TGFBl gene.

Preferably, the polymorphism pattern comprises a polymorphism in exon 1 of the TGFBl gene, preferably, a Arg25Pro polymorphism in exon 1 of the TGFBl gene. A first allele of the Arg25Pro polymorphism is a guanine base, and a second allele of the Arg25Pro polymorphism is a cytosine base.

Alternatively, or additionally, the polymorphism pattern may comprise a polymorphism in the promoter region of the TGFBl gene. Preferably, the polymorphism in the promoter region is at the -509 nucleotide position and is, more preferably, a thymine/cytosine (T/C) polymorphism. A first allele of the -509 nucleotide position is a cytosine base, and a second allele of the -509 nucleotide position is a thymine base.

According to the invention, an individual who carries either or both of the second alleles is classified as being at a higher risk for progression of renal disease when compared against an individual who has the first allele for the polymorphic regions. In this specific embodiment, the presence of either or both of the second alleles correlates with a susceptibility to renal failure.

Advantageously, this embodiment requires a low concentration of blood. However, other means for collecting DNA and determining polymorphism patterns which are known in the art may be used.

Preferably, the kit comprises DNA sampling reagents and, preferably, PCR amplification reagents. Preferably, the PCR amplification reagents comprise the use of Ampli-Taq Gold.

Preferably, said kit comprises at least one oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO .2 , SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

Oligonucleotide DNA primers that target the specific polymorphism DNA region within the genes of interest may be prepared so that, in the PCR reaction, amplification of the target sequences may be achieved. Said primers may comprise a detectable label.

Preferably, the kit comprises a control sample. Preferably, the control sample comprises one or more alleles selected from the group consisting of:- alleles of T(-509)C; and alleles of Arg25Pro.

The amplified DNA sequences from the target DNA may be analysed using restriction enzyme digestion to determine the polymorphism pattern present in the amplified sequences and thereby provide a genetic polymorphism profile of the individual.

The restriction enzyme digestion may comprise using the restriction enzyme _3su36l to digest the amplified DNA sequences when genotyping the individual for the T(-509)C polymorphism.

Preferably, said detecting comprises subjecting the amplified DNA to size analysis, preferably, electrophoresis and, preferably, comparing the results to a positive control and, preferably, a negative control.

According to a further aspect of the present invention there is provided a method of therapy comprising screening an individual for susceptibility to progression of renal disease and, if a genetic pre-disposition comprising a polymorphism pattern in TGFBl gene is identified, treating that individual to delay, reduce or prevent the progression of renal failure.

A suitable treatment to prevent, reduce or delay progression of renal failure may be hormone replacement therapy or gene therapy. The use of this therapy can thus be commenced in individuals likely to show a predisposition to the progression of renal disease.

According to a further aspect of the present invention, there is provided a method of treating an individual having a susceptibility to progression of renal disease, said method comprising the steps of:-

(i) determining the genotype of an individual to identify the presence of a polymorphism pattern in TGFBl gene, which polymorphism pattern is correlated with susceptibility to progression of renal disease; and (ii) administering to the individual a therapeutic agent that prevents, reduces or delays progression of renal disease.

According to a further aspect of the present invention, there is provided a method of identifying a polymorphism correlated with susceptibility to renal disease, said method comprising: -

(i) identifying in a cohort of individuals having a first polymorphism pattern of TGFBl gene, a second polymorphism pattern; and (ϋ) determining whether the second polymorphism pattern is correlated with the first polymorphism pattern of the TGFBl gene.

Other methods known in the art may be used to identify a further polymorphism correlated with susceptibility to progression of renal disease.

Preferably, the first polymorphism pattern is correlated with susceptibility to renal disease, preferably, susceptibility to progression of renal failure.

The first polymorphism pattern may comprise either/or the Arg25Pro or T(-509)C polymorphisms of TGFBl.

The second polymorphism pattern may be on the same gene as the first polymorphism pattern, but may be on any gene. Preferably, said determining comprises linkage disequilibrium analysis between said first and second polymorphism patterns.

According to a further aspect of the present invention, there is provided a method of identifying an allele associated with susceptibility to renal disease, said method comprising identifying an allele which is in linkage disequilibrium with either or both of Arg25Pro and/or T(-509)C polymorphisms in TGFBl gene, said polymorphisms being associated with susceptibility to renal disease.

According to a further aspect of the present invention, there is provided a method for determining a patient's susceptibility to renal disease, said method comprising: - (a) detecting a first allele of a first polymorphism of the TGFBl gene of a patient; and

(b) comparing the first allele of the first polymorphism to a second allele of a second polymorphism, (i) wherein the second allele is known to be associated with renal disease; and (ii) wherein correlation of the first allele with the second allele indicates susceptibility to renal failure.

According to a further aspect of the present invention there is provided a method for determining a patient's susceptibility to renal disease, said method comprising: (a) isolating DNA from a patient; (b) analysing said DNA to determine a first allele of a first polymorphism; and

(c) comparing said first allele with a second allele of a second polymorphism which is associated with renal disease, wherein correlation of said first allele with said second allele is indicative of susceptibility to renal disease.

Preferably, the second allele is selected from the group consisting of the Arg25Pro and T(-509)C polymorphisms of TGFBl.

According to a further aspect of the present invention there is provided a method of screening for progression of renal disease in a patient, the method comprising detecting the presence or absence of nucleic acid encoding a T-allele of a T(-509)C polymorphic region of TGFBl gene in said patient, wherein presence of the T-allele of the T(-509)C polymorphism in the TGFBl gene is indicative of progression of renal disease.

Preferably, the method further comprises the step of isolating genomic DNA from the patient.

According to a further aspect of the present invention there is provided a method of screening for progression of renal disease in a patient, the method comprising detecting the presence or absence of nucleic acid including a C-allele of a Arg25Pro polymorphic region at exon 1 of TGFBl gene in said patient, wherein presence of the C-allele of the Arg25Pro polymorphic region at exon 1 of the TGFBl gene is indicative of progression of renal disease.

Preferably, the method further comprises the step of isolating genomic DNA from the patient.

According to another aspect of the present invention, there is provided use of nucleic acid comprising SEQ. ID NO: 6 or 7 for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease. According to another aspect of the present invention, there is provided use of nucleic acid comprising a T(- 509) C polymorphism of TGFBl gene for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease.

According to another aspect of the present invention, there is provided use of a polypeptide comprising SEQ. ID NO: 8 or 9 for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease.

According to another aspect of the present invention, there is provided use of nucleic acid comprising SEQ. ID NO: 6 or 7 for drug research purposes for retarding or preventing the progression of renal disease.

According to another aspect of the present invention, there is provided use of nucleic acid comprising a T(- 509) C polymorphism of TGFBl gene for drug research purposes for retarding or preventing the progression of renal disease.

According to another aspect of the present invention, there is provided use of a polypeptide comprising SEQ. ID NO: 8 or 9 for drug research purposes for retarding or preventing the progression of renal disease.

Preferably, said drug research purposes comprises the generation of a molecular model of said nucleic acid or said polypeptide.

Preferably, the nucleic acid is isolated and, preferably, further comprises functional and/or structural variants thereof.

Preferably, the polypeptide is isolated and, preferably, comprises functional and/or structural variants thereof.

Preferably, and advantageously, the aspects of the present invention allow for the identification of an individual's genetic polymorphism pattern associated with progression to renal failure. Advantageously, the identification of those at risk allows preventative measures to be initiated prior to progression from CRF to ESRD.

Any one or more of the features described herein may be combined with any of the above aspects, except where said features are mutually exclusive or otherwise contradictory.

The present invention will now be described, by way of example, with reference to the accompanying drawings in which: -

Figure 1 shows an electrophoretic gel of PCR products for genotyping TGFBl Arg25Pro polymorphism in exon 1;

Figure 2 shows an electrophoretic gel for genotyping TGFBl promoter polymorphism at nucleotide position -509, after digestion of an initial PCR product with _5su36I;

To the sequence listing in which :-

SEQ ID NO.l is a first forward PCR primer for genotyping TGFBl Arg25Pro polymorphism of exon 1;

SEQ ID NO.2 is a second forward PCR primer for genotyping the TGFBl Arg25Pro polymorphism of exon 1;

SEQ ID NO.3 is a reverse PCR primer for genotyping the TGFBl Arg25Pro polymorphism of exon 1;

SEQ ID NO.4 is a forward PCR primer for genotyping the TGFBl promoter polymorphism at -509 nucleotide position;

SEQ ID NO.5 is a reverse PCR primer for genotyping the TGFBl promoter polymorphism at -509 nucleotide position;

SEQ ID NO.6 is a nucleic acid for the TGFBl G-allele of the Arg25Pro polymorphism of exon 1;

SEQ ID NO.7 is a nucleic acid sequence for the TGFBl C- allele of the Arg25Pro polymorphism of exon 1;

SEQ ID NO.8 is a protein sequence for the TGFBl G-allele of the Arg25Pro polymorphism of exon 1; and

SEQ ID NO.9 is a protein sequence for the TGFBl C-allele of the Arg25Pro polymorphism of exon 1.

Example 1

The aim of the research was to investigate the role of genetic variation in the transforming growth factor beta 1 gene (TGFBl) in progression of chronic renal failure (CRF) to end stage renal disease (ESRD) . Therefore, the research focussed on two polymorphisms in the TGFBl gene, i.e. Arg25Pro located in exon 1 of the TGFBl gene and, T-509C in the promoter of the TGFBl gene, in order to determine whether there was an association between these polymorphisms and the progression of renal failure.

The study was carried out on patients having chronic renal failure (CRF), i.e. renal patients, and on a control group selected from the normal population. Genotyping for the two alleles was carried out to determine whether there was:- a) any difference between the renal patients and the control group in allele frequencies or carriage rates, which would indicate that these markers influenced susceptibility to chronic renal failure, or b) any association between the two alleles and progressive and/or non-progressive renal failure, indicating that the markers might be useful in assessing risk of progression from CRF to end-stage renal failure (ESRF) .

Collection of Blood Samples

Ethical committee approval for this study was gained from the South Sheffield Ethics Committee, Northern General Hospital, Sheffield, UK and all patients gave informed consent. 140 samples were collected from patients with CRF.

Inclusion Criteria: Patients with creatinine concentration >150 μmol/1, white Caucasian, any age group, with and without dialysis treatment . Exclusion criteria:

Patients with creatinine concentration <150 μmol/1, non- Caucasian patients, relatives of patients included in the study.

Patients from whom blood was collected:

1. Blood samples collected from CRF patients:

a) Patients on chronic ambulatory peritoneal dialysis (CAPD) - these samples were collected from the kidney outpatient clinic at the Northern General Hospital, Sheffield. b) Patients on haemodialysis - these samples were collected from in-patients at the Kidney Institute at the Northern General Hospital, Sheffield. c) CRF patients from the out-patient renal clinic at the Northern General Hospital, Sheffield.

Patient details collected Patient's name, hospital number, research number, age, gender, weight, height, race, smoking, diet, diagnosis, date of diagnosis, other diseases, family history, renal biopsy, date of renal biopsy, hypertension, serum creatinine concentration, proteinuria, haematuria, serum cholesterol, serum triglyceride and treatment.

Depending on the creatinine slope, which was calculated by 1/creatinine against time, patients with CRF were classified into:- (i) End Stage Renal Failure (ESRF) - patients on renal replacement therapy such as dialysis; (ii) Fast Progressors (FP) - these patients have a creatinine slope of >-0.000035; (iii) Slow Progressors (SP) - these patients have a creatinine slope of <-0.000035; and (iv) Non Progressive (NP) - these patients have a creatinine slope of zero.

Furthermore, the patients were divided into whether the cause for CRF was either known or unknown (idiopathic) .

The data were collated using a Microsoft Excel spreadsheet and held confidentially, ie the patient names and hospital numbers were removed.

General Methods

Reactions and manipulations involving microbiological and molecular biological techniques, unless otherwise stated, were performed as generally described in Sambrook et al . , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) .

Ampli-Taq Gold used in some PCR reactions was from Roche Molecular Systems Inc. (Pleastanton, USA). Restriction enzymes were from Promega (Madison, USA) , and thermocyclers were from Biometra (Gottingen, Germany) .

DNA Extraction

10ml of blood was collected from each subject, anti- coagulated with EDTA and used for DNA extraction.

Buffers for DNA extraction:

1) Buffer A:

0.01M Tris Base, 0.32M Sucrose, 0.08M MgCl₂

The pH was adjusted to 8.0. The buffer was autoclaved and 10ml of Triton-X 100 was added.

2) Buffer B:

0.4M Tris Base, 0.06M EDTA, 0.150M NaCl₂ The pH was adjusted to 8.0. The buffer was autoclaved and SDS was added to give a final concentration of 1%.

3) 5M Sodium perchlorate

Protocol for DNA Extraction:

From Whole Blood:

After defrosting, each sample was poured into a 50ml Universal tube to which 40ml of buffer A was added. The samples were then centrifuged at room temperature (3000g for 15min) . The supernatants were discarded and the pellets were resuspended in Buffer A to a volume of 20ml. The samples were again centrifuged at room temperature (3000g for 15min) . The supernatants were removed and 1ml of Buffer B was added to each tube to resuspend the pellets. The contents were transferred to 1.5ml Eppendorf tubes to which 300μl of sodium perchlorate (5M) was added. The samples were mixed end-over-end several times and then micro-centrifuged for lOmin. 600μl of each supernatant was transferred to a clean Eppendorf tube and 700μl of ice cold chloroform was added. The samples were mixed end- over-end for 3min and were then micro-centrifuged for 10 min. The aqueous phase was transferred to a clean Eppendorf to which 2x the volume of ice-cold ethanol was added. The samples were mixed gently and then micro- centrifuged for 15min at 4°C. The supernatants were removed and the DNA was air dried for lOmin. The DNA pellet was resuspended in 200-500 μl of sterile distilled water. The optical density at 260nm was read to determine the concentration of the DNA extracted, and the ratio of absorbance at 260nm and 280nm was used to assess its quality. The degree of degradation of DNA was examined by agarose gel electrophoresis.

Polymerase Chain Reaction (PCR) : PCR was used to identify the alleles of the two polymorphisms in the TGFBl gene in all subjects and the control group. Blood samples were extracted using the methods previously described and PCR was used to analyse these samples.

1. Genotyping the TGFβ-1 polymorphism at codon 25 (Arg25Pro) within exon 1

Amplification Refractory Mutation System (ARMS) PCR was used to genotype the Arg25Pro polymorphism (Li, et al . , 1999) . Two forward (sense) primers, FI & F2, were designed to be identical to the sequence of the two alleles (G/C) over a region preceding and including the position of each variant nucleotide. One common reverse (antisense) primer, RI, was used for each PCR reaction (Li, et al . , 1998). If the forward primer matched with the allele, it bound to the complementary strand of the allele sequence, permitting amplification with this primer of a 196bp fragment. However, if the primer did not exactly match with the allele, amplification did not occur and no fragment was generated. Two reactions were performed for each DNA sample from each patient, each one containing one of the two forward primers and the common reverse primer. Therefore, it was possible to determine whether each sample was homozygous or heterozygous for each of the G and the C alleles.

The primer sequences which were used to detect the G/C alleles were:-

A standard 50μl PCR reaction mix and conditions were used: -

Standard 50μl PCR reaction mix: 35.5μl water, 5μl PCR buffer (as supplied by the manufacturer) , lμl MgCl₂ (ImM) , 0.5μl FI (SEQ ID NO.l), F2 (SEQ ID NO .2 ) & Rl (SEQ ID NO.3) primers (20pM) , 4μl dNTPs (0.2mM), 2μl template DNA (lOOng), 0.5μl Taq polymerase (2.5 units).

Negative controls for each reaction were used (the reaction mix as described above, except with 2μl of water instead of DNA) to detect possible contamination.

Standard PCR conditions: 1 cycle: 95°C for lOmin, 94°C for 5min; 5 cycles: 72°C for 60s, 60°C for 60s, 94°C for 60s; 30 cycles: 72°C for 30s, 56°C for 30s, 94°C for 30s; followed by 1 cycle: 72 °C for 5min. Following PCR, the products were separated by electrophoresis on a 2% (w/w) agarose gel at 80V for 40min

(room temperature) . To visualize DNA, gels were stained with ethidium bromide and viewed under ultraviolet light (see Figure 1) .

2. Genotyping the TGFBl promoter polymorphism at T(-509)C PCR was also used for genotyping the polymorphism at the promoter of the TGFBl gene. One forward (sense) primer, F3, and one reverse (antisense) primer, R2, were used for amplification of T/C polymorphism for each DNA sample. PCR produced a fragment of length 296bp (Grainger et al., 1999) .

The primer sequences used to detect the T/C alleles are:

A standard 50μl PCR reaction mix and conditions were used:

Standard 50μl PCR reaction mix: 35.5μl water, 5μl PCR buffer (as supplied by the manufacturer) , lμl MgCl₂ (I M) , 0.5μl F (SEQ ID NO.4) & R (SEQ ID NO.5) primers (20pM) , 4μl dNTPs (0.2mM), 2μl template DNA (lOOng) , 0.5μl Taq polymerase (2.5 units).

Negative controls for each reaction were used (the reaction mix as described above, except with 2μl of water instead of DNA) to detect possible contamination.

Standard PCR conditions: 1 cycle: 95°C for 5min; 34 cycles: 72°C for 60s, 60°C for 60s, 95°C for 60s; followed by 1 cycle: 72°C for lOmin.

Following PCR, buffer, unincorporated nucleotides, template DNA and enzyme from the PCR reaction were removed by ethanol precipitation. The PCR products were precipitated with three volumes of 96% (v/v) ethanol and a tenth volume of sodium acetate (3M, pH 5.2) . These were mixed and incubated for one hour at -20 °C. The DNA was pelleted by centrifugation (18000rpm for lOmin) , the supernatant was discarded and the DNA pellet was resuspended in 20μl of sterile distilled water. The DNA was then digested with Bsu36l in the following reaction mix: digest containing 10U Bsu36I at 37 °C for 12 hrs, in the buffer supplied by the manufacturer (David et al . , 1999). Following digestion, the products were separated by electrophoresis on a 1.5% (w/w) agarose gel at 80V for 40min (room temperature) . If the patient is homozygous for the C allele (CC) , the PCR product would not be digested and the band would be 296bp in length. If the patient is heterozygous (CT) then three bands would be produced, one at 296bp (for the C allele) and bands at 190bp and 106 bp (for the T allele) . The 106 bp band is often not visible under the conditions used. If the patient is homozygous for the T allele (TT) , the PCR products would be completely digested and a single band of 190bp would be visible. Results

The balance of genotypes observed conformed with Hardy- Weinberg expectations for the observed allele frequencies (analysed by χ² test) .

There was no significant difference between allele frequencies at these loci in the patient group and those in ethnically-matched controls, indicating that these alleles were unlikely to affect susceptibility to chronic renal failure. The data were further analysed to determine whether the alleles tested were associated with risk of progression to ESRF.

1) Results of Genotyping for TGFBl polymorphism Arg25Pro:-

Figure 1 shows an electrophoresis gel with the ARMS-PCR results of a selected number of individuals with idiopathic chronic renal failure (CRF) , whose samples have undergone polymorphism pattern (genotype) analysis for the alleles at Arg25Pro using the methods described above.

Two reactions were carried out for each subject typed, one to detect each possible allele, ie G and C alleles. Lanes 2 and 3 show amplification products from subject A, who is a C homozygote. Subject B (represented in lanes 3 and 4) is heterozygous, ie. Has the G and the C alleles at this position in the gene.

Patients were classified as "progressors" or "non- progressors" on the basis of 1/creatinine slope. Patients with end-stage renal failure were included in the "progressors" group. Since G allele homozygotes are rare, genotypes were grouped as (i) carrying (with G-allele) , or (ii) not carrying (without G-allele) at least one copy of the G allele.

Association with progressive disease :

χ² = 0.49 (p<0.05 with two degrees of freedom)

These data indicate that carriage of the G-allele is associated with a lower risk of progression to end-stage renal disease (ESRF) , and non-carriage of the G-allele with a higher risk. The odds ratio (approximate relative risk) equals 5.8 (95% confidence interval 1.7-19.4).

Further statistical analysis was carried out with the Microsoft Excel package. F-tests were used to test for significant differences in variance of proteinuria at onset, and mean proteinuria between patients carrying and not carrying the G-allele. A t-test (unpaired, two-tailed, for equal or unequal variance, as appropriate) was then applied to test for significant difference between means of these measurements.

Summary

The above data indicate that there is a significant association between the genotype at Arg25Pro of the TGFBl gene and:

1) the progression of idiopathic CRF to end stage renal failure (ESRF) ;

2) proteinuria, both at onset and mean proteinuria.

Therefore, the present invention provides a method of stratifying individuals according to risk of progression of CRF to ESRD.

2.Results of Genotyping for TGFBl promoter polymorphism T(-509)C

Figure 2 shows an electrophoresis gel of the PCR results which were subsequently digested with Bsu36I to determine the genotype at this polymorphic region. Lanes 2-11 each contain digested PCR products from a different subject. Primers used for this amplification were F3 and R2 in each case. Association with progression of idiopathic CRF:-

χ² = 6.5 (p<0.05 with two degrees of freedom).

These data indicate that carriage of the T-allele is associated with a higher risk of progression to end-stage renal disease (ESRF) , and non-carriage of the T-allele with a higher risk. The odds ratio (approximate relative risk) is 2.85 (with 95% confidence interval 1.2-6.8).

These data indicate that genotype at this locus is also associated with risk of progressive disease of CRF to ESRF. No associations were observed between the genotype and proteinuria at diagnosis, or mean proteinuria.

Linkage disequilibium Studies

Haplotype analysis indicates that there is significant linkage disequilibrium between alleles at the two loci studied. This analysis was carried out in all individuals where phase could be established unambiguously:

χ^z = 23 . 42 (with 2 degrees of freedom) p < 0 . 0001

Summary

The above data show that there is an association between the presence of the T-allele of the TGFBl promoter at T- 509C and the progression of CRF. Furthermore, haplotype analysis showed that the two alleles at codon 25, i.e the G-allele and the C-allele, and the two alleles at the promoter site, ie the T-allele and the C-allele are in strong linkage disequilibrium.

Therefore, the present invention provides a method of identifying individuals at risk to progression of CRF to ESRD. These data suggest that other polymorphic markers within, or close to this gene may also show significant linkage disequilibrium, and may also be useful in stratifying CRF patients for risk of progression to ESRF.

References

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The reader' s attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) , may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment ( s ) . The invention extend to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. A method for determining an individual's susceptibility to renal failure, said method comprising :- (i) obtaining a sample from an individual; and

(ii) detecting the presence of a genetic polymorphism pattern in TGFBl gene in said sample which polymorphism pattern is associated with renal failure .

2. A method according to claim 1, wherein said susceptibility to renal failure means the individual is susceptible to progression of renal failure.

3. A method according to either claim 1 or claim 2, wherein the polymorphism pattern comprises at least one polymorphism or polymorphic region of the TGFBl gene.

4. A method according to claim 3, wherein said polymorphism is selected from any of the following polymorphisms:- at position -988 (C/A) , -800 (G/A) , -509 (C/T) upstream of the coding region of the TGFBl gene, codon 10 (LeulOPro) of exon 1 of the TGFBl gene, codon 25

(Arg25Pro) of exon 1 of the TGFBl gene, codon 263 of exon 5 of the TGFBl gene, and a (C) insertion site at the 3' at position 72 of the non-translated region of said TGFBl gene.

5. A method according to either claim 3 or claim 4, wherein* the polymorphism pattern comprises a further polymorphism which is in linkage disequilibrium with the polymorphism which is known to be associated with renal failure.

6. A method according to either claim 4 or claim 5, any two of said polymorphic regions correlate with the progression of renal disease.

7. A method according to claim 6, wherein the first polymorphism comprises a first allele and a second allele.

8. A method according to either claim 6 or claim 7, wherein the second polymorphism comprises a first and a second allele.

9. A method according to either claim 7 or claim 8, wherein the second alleles of the first and the second polymorphic regions correlate with the progression of renal failure.

10. A method according to any preceding claim, wherein the polymorphism pattern comprises a polymorphism in exon 1 of the TGFBl gene.

11. A method according to any preceding claim, wherein the polymorphism pattern comprises an Arg25Pro polymorphism in exon 1 of the TGFBl gene.

12. A method according to claim 11, wherein the second allele of the Arg25Pro polymorphism is a cytosine base.

13. A method according to any of claims 1 to 9, wherein the polymorphism pattern comprises a polymorphism in the promoter region of the TGFBl gene.

14. A method according to claim 13, wherein the polymorphism in the promoter region is at the -509 nucleotide position.

15. A method according to either claim 13 or claim 14, wherein the polymorphism in the promoter region is a thymine/cytosine (T/C) polymorphism.

16. A method according to any preceding claim, wherein the sample comprises nucleic acid.

17. A method according to claim 16, wherein the nucleic acid encodes at least the TGFBl gene.

18. A method according to either claim 16 or claim 17, wherein the nucleic acid encodes at least the region upstream of the start codon of the TGFBl gene.

19. A method according to any preceding claim, wherein the sample comprises genomic DNA.

20. A method according to any preceding claim, wherein the sample comprises at least the 25^th codon of exon 1 and the -509 nucleotide region of the promoter of the TGFBl gene.

21. A method according to any preceding claim, wherein said detecting comprises PCR amplification of the sample.

22. A method according to any preceding claim, wherein said detecting comprises ARMS (amplification refractory mutation system) PCR of the sample when detecting for an Arg25Pro polymorphism.

23. A method according to any preceding claim, wherein said detecting comprises amplifying at least the region encoding codon 25 of the TGFBl gene and identifying the allele encoded by said amplified DNA.

24. A method according to any preceding claim, wherein said detecting comprises amplifying at least the promoter region of the TGFBl gene and identifying the allele encoded_^ by said amplified DNA.

25. A method according to any of claims 21 to 24, wherein the PCR amplification employs at least one primer comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO .4 and SEQ ID NO.5.

26. A method according to either claim 22 or claim 23, wherein the PCR amplification employs at least one primer comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO .2 and SEQ ID NO.3 when detecting for the Arg25Pro polymorphism.

27. A method according to claim 24, wherein the PCR amplification employs at least one primer comprising a sequence selected from either SEQ ID NO.4 or SEQ ID NO.5 when detecting for the T(-509)C promoter polymorphism.

28. A method according to any of claims 21-27, wherein said detecting comprises subjecting the amplified DNA to size analysis.

29. A method according to claim 28, wherein said size analysis is preceded by restriction enzyme digestion.

30. A method according to any of claims 21 to 28, wherein said detecting comprises digesting the amplified DNA with J3su36I, and then, subjecting the resultant digested DNA to electrophoresis.

31. A method according to any preceding claim, wherein said detecting comprises sequencing the DNA encoding the polymorphisms to determine the allele or alleles present.

32. A kit for identifying an individual's genetic polymorphism pattern in TGFBl gene which pattern i.s associated with renal disease, the kit comprising: -

(i) means for determining the presence of a genetic polymorphism pattern in TGFBl gene, wherein said polymorphism pattern is associated with progression of renal disease.

33. A kit according to claim 32, wherein the kit comprises DNA sample collecting means.

34. A kit according to either claim 32 or claim 33, wherein the polymorphism pattern comprises at least one polymorphism and/or polymorphic region of the TGFBl gene.

35. A kit according to any of claims 32 to 34, wherein the polymorphism pattern comprises an Arg25Pro polymorphism in exon 1 of the TGFBl gene.

36. A kit according to any of claims 32 to 35, wherein the polymorphism pattern comprises a polymorphism in the promoter region of the TGFBl gene.

37. A kit according to claim 36, wherein the polymorphism in the promoter region is at the -509 nucleotide position.

38. A kit according to any of claims 32 to 37, wherein the kit comprises DNA sampling reagents.

39. A kit according to any of claims 32 to 38, wherein the kit comprises PCR amplification reagents.

40. A kit according to any of claims 32 to 39, wherein said kit comprises at least one oligonucleotide comprising a sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO .4 and SEQ ID NO.5.

41. A kit according to any of claims 32 to 40, wherein the kit comprises a control sample.

42. A kit according to claim 41, wherein the control sample comprises one or more alleles selected from the group consisting of:- alleles of T(-509)C; and alleles of Arg25Pro.

43. A method of therapy comprising screening an individual for susceptibility to progression of renal disease and, if a genetic pre-disposition comprising a polymorphism pattern in TGFBl gene is identified, treating that individual to delay, reduce or prevent the progression of renal failure.

44. A method of treating an individual having a susceptibility to progression of renal disease, said method comprising the steps of:-

(i) determining the genotype of an individual to identify the presence of a polymorphism pattern in TGFBl gene, which polymorphism pattern is correlated with susceptibility to progression of renal disease; and

(ii) administering to the individual a therapeutic agent that prevents, reduces or delays progression of renal disease.

45. A method of identifying a polymorphism correlated with susceptibility to renal disease, said method comprising: -

(i) identifying in a cohort of individuals having a first polymorphism pattern of TGFBl gene, a second polymorphism pattern; and (ii) determining whether the second polymorphism pattern is correlated with the first polymorphism pattern of the TGFBl gene.

46. A method of identifying an allele associated with susceptibility to renal disease, said method comprising identifying an allele which is in linkage disequilibrium with either or both of Arg25Pro and/or T(-509)C polymorphisms in TGFBl gene, said polymorphisms being associated with susceptibility to renal disease.

47. A method of screening for progression of renal disease in a patient, the method comprising detecting the presence or absence of nucleic acid encoding a T-allele of a T(- 509) C polymorphic region of TGFBl gene in said patient, wherein presence of the T-allele of the T(-509)C polymorphism in the TGFBl gene is indicative of progression of renal disease.

48. A method of screening for progression of renal disease in a patient, the method comprising detecting the presence or absence of nucleic acid including a C-allele of a Arg25Pro polymorphic region at exon 1 of TGFBl gene in said patient, wherein presence of the C-allele of the Arg25Pro polymorphic region at exon 1 of the TGFBl gene is indicative of progression of renal disease.

49. Use of nucleic acid comprising SEQ. ID NO: 6 or 7 for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease.

50. Use of nucleic acid comprising a T(-509)C polymorphism of TGFBl gene for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease.

51. Use of a polypeptide comprising SEQ. ID NO: 8 or 9 for the preparation of a medicament for the treatment of retarding or preventing the progression of renal disease.

52. Use of nucleic acid comprising SEQ. ID NO: 6 or 7 for drug research purposes for retarding or preventing the progression of renal disease.

53. Use of nucleic acid comprising a T(-509)C polymorphism of TGFBl gene for drug research purposes for retarding or preventing the progression of renal disease.

54. Use of a polypeptide comprising SEQ. ID NO: 8 or 9 for drug research purposes for retarding or preventing the progression of renal disease.