GB2534879A

GB2534879A - The identification of genomic markers in the first intron of the LEPREL 1 gene which predict resistance or susceptibilty to fibrotic disorders

Info

Publication number: GB2534879A
Application number: GB1501727.0A
Authority: GB
Inventors: Stratton Richard; Lopez Henry; Martin George
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-02-03
Filing date: 2015-02-03
Publication date: 2016-08-10
Also published as: GB201501727D0

Abstract

A quantitative PCR method was used to genotype for the presence of a copy number variation (CNV) present in the first intron sequence of the LEPREL1 gene which encodes a prolyl 3- hydroxylase involved in collagen biosynthesis and an association with firbrotic diseases was established. In particular these CNVs are in Transforming growth factor beta (TGFb) enhancer sites. Identification of these CNVs is suggested as a marker for diagnosis and prognosis, including identifying patients as drug responders, for a range of scarring and fibrosis conditions including chronic kidney disease, pulmonary fibrosis, cardiac failure, and scleroderma.

Description

Provisional Patent Application THE IDENTIFICATION OFGENOMIC MARKERS IN THE FIRST INTRON OF THE LEPREL 1 GENE WHICH PREDICT RESISTANCE OR SUSCEPTIBILITY TO FIBROTIC DISORDERS

Description

Fibrosis is a common response to trauma, infection, chemotherapy, radiation, metabolic diseases such as diabetes and has a role in certain solid cancers and even aging. In fibrotic reactions, inflammation often initiates a wound healing-like response. A variety of cells enter the damaged area to remove contaminating debris and these in turn attract fibroblastic cells to penetrate into the wound where they are stimulated to proliferate and deposit collagen and other connective tissue proteins. While this is beneficial in the case of wounds, the replacement of normal tissue components and cells by scar tissue adversely alters normal tissue structure and function most notably in the lungs, the kidneys, the heart, the vasculature and other body sites. In some disorders such as scleroderma, the fibrotic reactions are progressive, moving along the skin and to internal organs affecting the lungs, GI tract and kidneys. Some individuals are prone to fibrotic disorders while others are not. The reasons for this variation from individual to individual were not known until now. Here, we reveal that the presence of certain copy number variations (CNVs) in the first intron of the LEPREL 1 gene sensitize the bearer to develop fibrotic reactions. However, if the DNA sequences are absent from an individual's DNA they are protected against fibrosis. When the DNA sequences are present, a critical enzyme in collagen synthesis is abnormally elevated and is even higher when exposed to TGF(3, a pro-fibrotic cytokine strongly implicated in scarring and fibrosis. Thus trauma or environmental factors can activate a sustained fibrotic response in individuals bearing these genetic markers. The information revealed here allows the development of diagnostics to assess the individual risk for developing a fibrotic disorder, particularly those caused by agents such as listed above as well as providing a target for development of therapeutics.

Background

Tissues are remarkably designed for their function. The lungs expand and contract and gases pass readily through them to the blood. If the lungs become fibrotic, both gas exchange and lung expansion are compromised and may even be incompatible with survival. Such side effects are common after radiation of the lungs in lung cancer treatment, as well as damage to the esophagus which can impair swallowing. The ability to identify those individuals who are particularly at risk of an excessive reaction to tissue damage would help to define more beneficial therapies as well as avoid adverse reactions. The invention described here enables one knowledgeable in the art to develop diagnostics. Moreover, there are no effective and safe therapeutics for treating fibrotic conditions. We describe a target which is readily druggable and its inhibition is relatively safe for normal tissue functions.

As a well-known example of a fibrotic disease, we studied DNA from patients with scleroderma (systemic sclerosis) and compared these to people with no evidence of scleroderma or other fibrotic disorder. Specifically, we examined a variety of genes for CNVs in the first intron. CNVs are sequences in the genome of individuals which can be new or inherited variants of 1000 nucleotides or more that can be duplicated or deleted in some individuals. Prior work has shown that such CNVs are associated with susceptibility or in some cases resistance to various disorders, but not previously known in the case of fibrotic disorders. In our survey we limited our examination to CNVs in the first intron of a number of genes of potential relevance to the disorder. Specifically, we examined CNVs in the first intron of genes involved in collagen synthesis, genes involved in estrogen action, and genes involved in inflammation. The first introns of genes often contain regulatory elements which enhance gene expression.

Scleroderma is classified as an autoimmune disorder since many patients have circulating antibodies directed to their own proteins. However, the most visible manifestation is a progressive fibrotic reaction affecting the skin and certain internal organs. While scleroderma is not directly inherited, the relative risk of scleroderma in first degree relatives of scleroderma patients is 13 time higher than in the general population. The current hypothesis is that there are genetic susceptibility factors triggered by environmental factors. Here we describe a genetic susceptibility factor for the disease.

Mechanisms in collagen synthesis and fibrosis Tissue injury from diverse internal and external processes activates potent responses. Physical damage elicits the clotting of blood which releases various cytokines from platelets and activates inflammation and a wound healing response. Cytokines such as the platelet derived growth factor (PDGF) play a dual role attracting fibroblasts to the wound site and stimulating their proliferation. Cytokines such as those of the TGF13 family induce the transcription of a variety of genes including those which code for collagen and those enzymes required for the formation, deposition and crosslinking of collagen molecules and fibers. Some 28 genetically distinct collagens have been identified with collagens 1 and 3 the most abundant in fibrotic tissues. The synthesis of collagen is a complex process. The collagen 1 and 3 genes are large and divided into many exons separated by non-coding introns. The first intron in the gene coding for procollagen al and a2 chain contains enhancers which are involved in the regulation of transcription of the genes. During and following transcription, the procollagen chains undergo association in a 2proal to 1proa2 ratio established by interaction via their carboxyl terminal domains. Several enzymatic modifications occur to the chains. 100 prolines in each chain in the sequence gly-X-proline where X stands for a number of possible amino acids, are 4-hydroxylated by prolyl 4-hydroxylase. These modifications are known to enhance the stability of the collagen triple helix. Certain lysines are converted by lysyl hydroxylase to hydroxylysine residues and then enzymatically glycosylated. Prolyl 4-hydroxylase and lysyl hydroxylase have the same cofactor requirements, namely ferrous iron, a-ketoglutarate, ascorbic acid, and oxygen.

There is an additional enzyme modification. One proline in the helical sequence near the carboxyl terminus in each chain in the sequence glycine-proline-Xis hydroxylated in the 3 position. Mutations altering this reaction lead to serious forms of osteogenesis imperfecta. One concept for the function of this modification is that prolyl 3-hydroxylase is a chaperone protein required to align the 3 chains so that the triple helix which forms is precisely aligned. This enzyme has the same cofactor requirements as those stated above namely, a-ketoglutarate, iron, ascorbic acid and oxygen. The 3 chains when assembled into a triple helical molecule, procollagen are then released from the cell. Specific proteolytic enzymes cleave portion of the molecule from both ends converting it to collagen which is deposited in fibers and cross-linked by lysyl oxidase.

Our discovery suggests that prolyl 3-hydroxylase expression is elevated in cells from scleroderma lesions which overproduce collagen due to certain variations in the sequence of their DNA which affect the expression of the enzyme.

Findings It is well established that fibroblast cultured from the active skin lesion of scleroderma patients produce more collagen than fibroblasts from normal skin and maintain this differential activity when passaged in culture, suggesting an epigenetic modification. The current hypothesis is that excessive collagen synthesis is driven by exposure of the cells to TGFP (Transforming Growth Factor beta) and that continued exposure maintains high collagen synthesis and leads to modifications in the DNA in cells continually exposed to TGFp so that they are fixed in this process.

We used QPCR (quantitative polymerize chain reaction) technology to assess CNVs in the first intron of some 33 genes, studying DNA samples purified from the blood of scleroderma patients and healthy controls. This technology allowed us to quantitate the CNVs as to no CNVs in either allele, one allele with a CNV or both alleles with CNV (Figure 1). As shown in the table one gene had CNVs of altered frequency in DNA from patients with scleroderma. These were CNVs found in the first intron of the LEPRL1 gene, an enzyme required for collagen synthesis. Moreover, this sequence contained binding sites for the SMAD proteins which are the nuclear activators induced by TGFP binding to the TGFP receptor. Furthermore analysis by us of single nucleotide polymorphisms (SNPs) from a large set of scleroderma and control DNA samples, revealed that an SNP adjacent to the CNV in the first intron of the LEPREL1 gene was also associated with the development of scleroderma, confirming the CNV data and explaining its mechanism (Table 1, Figure 4). Basal levels of the LEPREL1 enzyme protein product were higher in cells from the scleroderma patients (Figure 2) and elevated further by the addition of TGFP (Figure 3). Both the basal and TGFP induced levels of LEPREL1 were significantly higher in the scleroderma cells than in comparable cells from controls.

Invention We have identified a genomic marker in LEPREL1 that identifies individuals at risk of developing a fibrotic disease, scleroderma. However, it is likely to be a genomic marker for a variety of other fibrotic disorders such as idiopathic pulmonary fibrosis, kidney fibrosis, cardiac fibrosis, radiation caused fibrosis, and chemotherapy induced fibrosis and fibrosis occurring in certain aged individuals. Numerous ways to assess the genomic risk will be apparent to one skilled in the art including assessing CNV occurrence in patients DNA, mRNA levels by a variety of methods and even protein levels of prolyl 3-hydroxylase by immunological or activity assays.

Similarly, the excessive enzyme can be suppressed by any number of ways known to one knowledgeable in the art to develop safe and effective therapeutics.

Data supporting the claims Male Normal 9/50 20150 21150 Male SSc 1/48 18/48 29/48 Figure 1 Relative frequency of CNV in intron 1 of the LEPREL1 gene in scleroderma (SSc) and control DNA samples. Double deletion of the CNV (-/-) was associated with reduced risk of disease in males (p<0.044).

Ratios of LEPREL1 CNV Genotypes Male Normal Male SSc (-It) 0.18 0.40 0.38 \ 0.02 0.42 0.60 LEPREL1 (P3H2) expression in normal and scleroderma fibroblasts en N r4 ell d-i 1 2 3 4 5 6 7 8 9 10 EC EP cells Control Scleroderma Figure 2 Increased LEPREL1 production by cells cultured from scleroderma fibrotic lesions. Fibroblast cultures were established from scleroderma and healthy volunteer skin biopsies, and secreted proteins measured by Western blot of the conditioned media. Scleroderma fibroblasts (SScl-4) secreted much higher amounts of LEPREL1 protein into the media compared to control cells (NF1-3). Endothelial cell (HMEC-1) and epithelial cell (AS49) cultures were studied for comparison.

LEPREL1 (P3H2) expression in normal and scleroderma fibroblasts with and without TGFD treatment Figure 3 LEPREL1 protein levels increase following exposure of fibroblasts to TGFI3. Scleroderma and normal control fibroblasts were cultured with or without the addition of TGFI3 4ng/ml. Following 24 hour further culture media were removed and assayed by Western blotting for the presence of LEPREL1 protein. LEPREL1 protein levels were higher in scleroderma cultures and induced further following treatment with TGFI3.

EC EP cells Control Scleroderma 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a-I rsl

Z Z Z

LL LL LL 0 el (V rn

LL LL LL 0 0 lN

el (NI M

U

VI V) VI VI Li) VI V) VI V) VI In VI SNP Observed allele frequency Expected allele rs7612998 Healthy Scleroderma frequency Chi sq p Healthy Scleroderma 0.145318 CC 583 662 590.3017 654.6983 CT 77 68 68.75 76.25 TT 0 2 0.948276 1.051724 rs1447936 TT 391 352 387.4688 355.5312 0.586055 CT 228 209 227.8921 209.1079 CC 24 29 27.63909 25.36091 rs696065 AA 346 342 349.0706 338.9294 0.874194 AG 256 240 251.6555 244.3445 GG 52 53 53.27386 51.72614 rs1018343 GG 361 317 343.1531 334.8469 0.010309 AG 257 300 281.9119 275.0881 AA 43 28 35.93492 35.06508 Table 1 SNP analysis of scleroderma and control DNA samples. Known single nucleotide polymorphisms (SNPs) were selected for strategic location in the LEPREL1 gene choosing loci from the promotor region (rs7612998), the first intron (rs1447936 and rs1018343), as well as the 3' untranslated region (rs696065). Of these only rs1018343, which is in close proximity to the CNV studied, showed an association with scleroderma disease development (minor allele AA homozygous protective against scleroderma p<0.01).

LEPREL1 protein EXCESSNE CO....

CROSS I.: G OS

STABLE FIBROTIC LESIONS

ESTABLISHED FIBROSIS

Figure 4 Overall schematic demonstrating mechaniiim of action of LEPREL1 CNV in fibrosis. The presence of homozygous deletion (-/-) in the CNV in the first intron of the LEPRELl gene prevents the normal induction of LEPREL1 by pro-fibrotic growth factors. Consequently downstream alignment and cross linking of collagen is reduced and stabilization of fibrotic tissue is attenuated leading to reduced susceptibility of LEPREL1 CNV genotype -/-individuals to fibrosis.

Pro-fibrotic growth factor LEPREL1 gene N-terminal

Claims

Claims What is claimed is: 1. Identification of a genomic marker for fibrosis susceptibility, LEPREL1 CNVs.
2. Identify the LEPREL1 CNVs as a TGFp. enhancer site.
3. Methods for assessing the presence of the enhancer as well as for testing its function.
4. Identified a therapeutic target for therapeutic treatment development to avoid or reduce fibrosis.
5. Identified a diagnostic marker for assessing the potential hazard to the administration of potential fibrosis initiators such as surgery, radiation, or chemotherapy.
6. A method to discriminate between drug-responders vs drug non-responders using the status of CNV polymorphisms of claim #1 as a marker for patients' ability to respond to a certain drug among patients with a disease or condition that is associated with CNV polymorphisms of claim 1, comprising the steps of the identification, in prospective or retrospective setting, of drug responders among patients treated with said drug, and identifying the association between drug response and said polymorphism status.
7. A method of claim #1, wherein the homozygous presence of said polymorphism is used as disease or condition susceptibility marker in combination with other markers.
8. A method for treating or preventing the development of a disease or condition that is associated with CNV polymorphisms of claim #1, comprising the steps of detecting the status of said polymorphisms in the patient and choosing the drug known to be efficient in patients with this particular status of said polymorphisms