ES2537052B1 - Procedure for the prediction of non-viral alcoholic liver cirrhosis in a subject - Google Patents

Abstract

The present invention is a method for the prediction of non-viral liver cirrhosis in a subject, which comprises the detection of the presence of the KIR2DS5 gene in a biological sample obtained from said subject, the comparison of the result of the previous detection with a control and the Evaluation of their genetic predisposition to develop non-viral liver cirrhosis from the previous comparison. The method is a very useful tool to define a risk factor that allows to predict if a subject that consumes alcohol will have an inadequate immune response to protect against the development of fibrosis and its drift towards alcoholic cirrhosis not associated with viral infection.

Description

PROCEDURE FOR THE PREDICTION OF NON-VIRIC ALCOHOLIC HEPATIC CIRROSIS IN A SUBJECT

TECHNICAL SECTOR 5

The invention falls within the medical field of the diagnosis and prognosis of alcoholic cirrhosis in human patients with ethanolic habit without concomitant viral infection, associated with the prevention of the disease.

 10

BACKGROUND OF THE INVENTION

Immunoglobulin-like natural killer cell (NK) receptor (KIR) genes have recently been related to the response to bone marrow transplantation, assigning them a role in the anti-leukemic effect (Port F, Fischer JC, Uhrberg M. “The role of KIR genes and ligands in leukemia 15 surveillance.” Front Immunol. 2013. 7; 4:27). Also, with some types of organ transplants and predisposition to hepatitis C virus infections or autoimmune diseases (Zhou H et al., “Donor selection for killer immunoglobulin-like receptors B haplotype of the centromeric motifs can improve the outcome after HLA-identical sibling hematopoietic stem cell transplantation. ”Biol Blood 20 Marrow Transplant. 2014; 20 (1): 98-105).

KIRs are a group of membrane receptors encoded by a family of polymorphic genes located within the Leukocyte Receptor Complex (CSF) of chromosome 19. These genes encode membrane receptors with function of both inhibitors and activators. They are expressed on the surface of NK cells and in subpopulations of Tγ and Tδ lymphocytes. KIR receptors modulate the activity of NK or T cells through interaction with their specific ligands that are class I HLA molecules (Vilches C et al. “Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments, Tissue Antigens, 2007; 70 (5): 415-3022).

The KIR2DS5 activator gene has recently been linked to susceptibility to rheumatoid arthritis (Chandran V et al. "Killer-cell immunoglobulin-like receptor gene polymorphisms and susceptibility to psoriatic arthritis." Rheumatology (Oxford). 2014; 35

53 (2): 233-9, as opposed to protection against thrombocytopenia of immune origin. There is no known ligand for the receptors encoded by this gene in the art (Nowak I et al. "Does the KIR2DS5 gene protect from some human diseases?" PLoS One. 2010.26; 5 (8). Nor have KIR genes been related. with the ethanolic habit that leads to alcoholic cirrhosis.

Maintained alcohol intake over time results in liver fibrosis, which precedes cirrhosis. If at this fibrotic stage the patient stops drinking alcohol, the liver starts different regeneration mechanisms, while if the consumption persists the process is chronicled and can only be treated by liver transplantation. In short, alcoholic liver damage can only be reversed with ethanolic abstention in the initial stages. At present, alcoholic cirrhosis is diagnosed by the patient's history and statement about habitual alcohol consumption, physical examination, biochemical tests (bilirubin, AST / ALT, GGT, etc.), by imaging tests (Fibroscan) and above all by liver biopsy as a reference method to determine the degree of fibrosis. The great disadvantage of the biopsy is that it is an invasive technique. All these techniques serve only for the diagnosis once cirrhosis is established and none of them implies an early risk factor that can be used to prevent the disease. twenty

On the contrary, the method of the present invention does identify KIR2DS5 as a risk factor for susceptibility to alcoholic cirrhosis not associated with viruses, whose detection can be used to recommend ethanolic abstention.

 25

Hepatitis C virus can also lead to cirrhosis, but in this case the diagnosis is made by the presence of the virus and the quantification of the viral load by immunoassay. This method is not applicable to the diagnosis of alcoholic cirrhosis not infected by viruses. In some patients, cirrhosis due to virus C is also associated with alcohol consumption but not with KIR2DS5. Thus, the present invention 30 serves to discriminate both types of cirrhosis precisely because KIR2DS5 does not re-associate alcoholic cirrhosis caused by viral infection but is a factor highly associated with purely alcoholic cirrhosis without concomitant viral infection, as shown in the present application. .

 35

The problem of the technique is to find a biomarker of alcoholic liver cirrhosis not associated with viruses, with reliable predictive value and non-invasive detection. The solution proposed by the present invention is determination of the presence of the KIR2DS5 gene in the patient's genome.

 5

DESCRIPTION OF THE INVENTION

The present invention is a method for the prediction of non-viral alcoholic liver cirrhosis in a subject, comprising the detection of the presence of the KIR2DS5 gene in a biological sample obtained from said subject, the comparison of the result of the previous detection with a control and the evaluation of its genetic predisposition to develop the disease from the previous comparison. In a more restrictive aspect, the invention consists in the above procedure.

The invention is able to predict that KIR2DS5 is a determining risk factor 15 of the probability of development of non-viral alcoholic cirrhosis, produced only by alcohol consumption and independent of the intervention of other pathological factors, such as its association with hepatitis produced by viral infections.

In a preferable aspect of the process of the invention, said biological sample is a blood sample. In another preferable aspect it is a tissue sample, and in a more preferable aspect it is a saliva sample. Another very preferable aspect of the invention is that said subject is a male subject.

Functionally, a specific ligand for KIR2DS5 is not yet known, but given the effect observed in this pathology it can be assumed that this receptor is dependent on the existence of a ligand that can be overexpressed in liver cells involved in the progression of fibrosis, and capable to mediate the protective role of activated NK cells against liver fibrosis. This role would only be attributable to the action of the KIR2DS5 gene among the activator genes, whose action is also not affected by the concomitant presence of other KIR inhibitor genes. These KIR inhibitor genes not only are not associated with the predisposition to suffer from the disease but may even be protective elements against it, as is the case with KIR2DL2.

 35

An analysis of the KIR gene profile inhibitors and activators of healthy individuals and patients has been conducted to determine the frequency of the KIR2DS5 activator gene in the sick population.

Table 1 shows the frequencies of KIR genes for the total of 5 male patients included in the study and for the total of healthy male individuals used as a control group, as well as the results of the univariable analysis of the presence of the genes studied in the genotype of the patients and controls. It is observed that KIR2DS5 is the only KIR gene that is associated with alcoholic cirrhosis of non-viral origin in a highly significant way. It behaves as a high risk factor for the suffering of said disease since it indicates a predisposition to suffer it in subjects who are carriers of said gene. In addition to the presence of this gene and independently, the only variable that is also able to positively influence the development of the disease is age. In contrast and in contrast to KIR2DS5, the presence of the KIR2DL2 gene is negatively associated with the disease. fifteen

 Table 1. Frequency of KIR genes in male patients with non-viral and viral alcoholic cirrhosis, and in healthy controls that do not develop the disease.

 Patients with alcoholic cirrhosis

      Healthy Controls N = 319 Total patients N = 281 No viral infection N = 213 Viral infection N = 68

 KIR genes *

 P / A n (%) n (%) P1 n (%) n (%) P2 P3 P4

 iKIRs

 2DL1 (S1-)

 + 197 (61.8) 160 (56.9) 0.244 115 (54.0) 45 (66.2) 0.088 0.581 0.092

 - 122 (38.2) 121 (43.1) 98 (46.0) 23 (33.8)

 2DL2

 + 202 (63.3) 149 (53.0) 0.013a 112 (52.6) 37 (54.4) 0.015d 0.173 0.889

 - 117 (36.7) 132 (47) 101 (47.4) 31 (45.6)

 2DL3

 + 279 (87.5) 249 (88.6) 0.707 190 (89.2) 59 (86.8) 0.586 0.842 0.661

 - 40 (12.5) 32 (11.4) 23 (10.8) 9 (13.2)

 3DL1

 + 304 (95.3) 268 (95.4) 1,000 201 (94.4) 67 (98.5) 0.689 0.325 0.200

 - 15 (4.7) 13 (4.6) 12 (5.6) 1 (1.5)

 2DL5

 + 170 (53.3) 158 (56.2) 0.511 121 (56.8) 37 (54.4) 0.477 0.894 0.780

 - 149 (46.7) 123 (43.8) 92 (43.2) 31 (45.6)

 aKIRs

 2DS1 (L1 +)

 + 119 (37.3) 119 (42.3) 0.211 96 (45.1) 23 (33.8) 0.087 0.678 0.121

 - 200 (62.7) 162 (57.7) 117 (54.9) 45 (66.2)

 2DS2 (L2 +)

 + 201 (63.0) 146 (52) 0.006b 109 (51.2) 37 (54.4) 0.007e 0.217 0.677

 - 118 (37.0) 135 (48) 104 (48.8) 31 (45.6)

 2DS3

 + 107 (33.5) 93 (33.1) 0.931 65 (30.5) 28 (41.2) 0.508 0.263 0.107

 - 212 (66.5) 188 (66.9) 148 (69.5) 40 (58.8)

 2DS4

 + 305 (95.6) 266 (94.7) 0.704 199 (93.4) 67 (98.5) 0.323 0.486 0.128

 - 14 (4.4) 15 (5.3) 14 (6.6) 1 (1.5)

 2DS5

 + 86 (27.0) 99 (35.2) 0.033c 85 (39.9) 14 (20.6) 0.002f 0.360 0.004g

 - 233 (73.0) 182 (64.8) 128 (60.1) 54 (79.4)

 3DS1

 + 129 (40.4) 132 (47.0) 0.117 105 (49.3) 27 (39.7) 0.050 1,000 0.209

 - 190 (59.6) 149 (53.0) 108 (50.7) 41 (60.3)

P, presence; A, absence; N, total number of subjects; n, number of subjects with presence or absence of KIR genes. Comparisons have been made by Fisher's exact two-tailed test *, constitutive genes and pseudogenes have not been included. P1, P value obtained from the

comparison between patients with total alcoholic cirrhosis versus total healthy donors; P2 and P3; P values obtained from the comparison between patients with non-viral alcoholic cirrhosis and patients with viral alcoholic cirrhosis versus healthy controls, respectively; P4, P value obtained from the comparison of patients with non-viral alcoholic cirrhosis versus viral alcoholic cirrhosis. a, OR = 1,530; 95% CI: 1,103-2,120, P = 0.013; b, OR = 1,575; 95% CI: 1,137-2,183, 5 P = 0.006; c, OR = 0.679; 95% CI: 0.479-0.961, P = 0.033; d, OR = 1,557; 95% CI: 1,095-2,215, P = 0.015; e, OR = 1,625; 95% CI: 1,143-2,311, P = 0.007; f, OR = 0.556; 95% CI: 0.384-0.804, P = 0.002; g, OR = 2,561; 95% CI: 1,339-4,900, P = 0.004.

 10

A complementary analysis to that of Table 1 has also been carried out, aimed at evaluating the total telomeric genes in relation to the predisposition to suffer from the disease (Figure 1). In this case, an increase in the frequency of non-viral cirrhosis is only observed when KIR2DS5 is carried by the patient either alone or accompanied by any of the other two activating genes (KIR2DS1 and KIR3DS1), 15 or both. This suggests that these two genes are not in themselves risk factors but present an imbalance of binding with KIR2DS5. The prevailing effect is that of KIR2DS5 since it exceeds that observed for the other two genes in non-viral cirrhosis. In short, the result confirms that only the KIR2DS5 gene is independently associated with non-viral alcoholic cirrhosis as the responsible factor. twenty

Table 2 complements the data represented in Figure 1. This table shows the combined frequency of all KIR genes in the population of male patients with alcoholic cirrhosis (n = 281) and the binding imbalances of the KIR2DS5 gene with the rest. of KIR activator and inhibitor genes located in centromeric and telomeric regions of chromosome 19.

 30

 Table 2. Analysis of the union imbalance between the different KIR genes in the population of male patients with alcoholic cirrhosis.

 Centromeric Part

    Telomeric part

 3DL3

 52 89 53 56 33 98 98 100 100 95 47 35 42 95 0

 2DS2 41 99 70 72 50 50 52 52 50 59 57 58 49 52

 2DL3 42 47 28 91 92 89 88 86 48 40 43 85 89

 2DL2 71 71 51 51 53 53 51 60 56 58 50 53

 2DL5 77 56 56 56 56 52 87 77 84 51 56

 2DS3 36 36 33 33 34 64 56 64 34 33

 2DL1 100 98 98 95 47 36 42 93 98

 2DP1 98 97 96 47 35 42 94 98

 3DP1 100 95 47 35 42 95 100

 2DL4 95 47 35 42 94 100

 3DL1 42 31 38 100 95

 3DS1 87 94 42 47

 2DS5 89 30 35

 2DS1 37 42

 2DS4 95

 3DL2

Table 3 shows the result of the multivariable logistic regression analysis applied to confirm the associations described in the univariate analysis performed in healthy controls and patients with alcoholic cirrhosis. In accordance with all of the above, the only statistically significant variables associated with non-viral cirrhosis are the presence of the KIR2DS5 gene as predisposing to the development of the disease and that of KIR2DL2, which contrasts with the previous gene because it is a factor associated with the absence of disease The value of the age variable is the only one that is not confirmed with the multivariate analysis, which reinforces the role of KIR2DS5 as a risk factor.

 10

Table 3. Multivariate analysis of the main variables contemplated in the study.

 Variables

 OR (95% CI) P

 Age

 1,010 (0.998-1.022) 0.098

 KIR2DS5

 1,519 (1,066-2,164) 0,021

 KIR2DL2 KIR2DL3

 0.612 (0.432-0.666) 0.648 (0.560-1.437) 0.006 0.539

 In all cases, comparisons were made between the patient group and the control group as the reference group.

The method of the present invention allows predicting the susceptibility of a subject to suffer from the disease, or on the other hand its innate resistance against the harmful effects induced by alcohol consumption. Therefore, the result of its application serves to guide the need or not to continuously monitor individuals with a possible imbalance due to alcohol consumption, which is the primary cause of the disease. The objective is to serve as a guide for the prevention and advice about healthy habits and alcohol-free diets in the patient 20 susceptible to developing the disease.

The method of the invention is configured as a very useful tool for defining a risk factor that allows predicting whether a subject who consumes alcohol will have an inadequate immune response to protect against the development of fibrosis, and its drift towards the stage more advanced liver damage that represents alcoholic cirrhosis not associated with viral infection.

The invention also offers guidance on the individual genetic characteristics that favor the existence of an innate or natural resistance to suffering from this disease. The lack of predisposition of a subject to suffer from alcoholic cirrhosis could be associated with a better ability to produce an ideal immune response to maintain homeostatic balance and the state of health of the organism. Thus, even after any occasional immune imbalance you could recover your health status more easily.

It is possible that the protein molecule or molecules encoded by the KIR2DS5 gene modulate the activation of cytotoxic NK and T cells. This mechanism seems essential to favor fibrosis and / or liver cirrhosis because it can mediate by preventing the activation of star cells and their fibrotic transformation.

The molecular analysis method used in the PCR of the present invention offers early prediction of individual susceptibility to alcohol consumption and therefore the predisposition to suffer from alcoholic cirrhosis in subjects carrying the KIR2DS5 gene. The procedure is a quick and simple and non-invasive system (Table 4) that can be performed with biological samples of non-surgical extraction in the populations most exposed to alcohol damage. twenty

In a preferable aspect of the process of the invention, the detection of the KIR2DS5 gene in the patient's biological sample comprises a polymerase chain reaction (PCR). The primers developed in the present invention (Table 4) are located in exon 4 encoding the extracellular domain D1 of the KIR2DS5 25 gene (Figure 2), and are capable of specifically amplifying any of the alleles identified to date for this gene, as shown in Table 5. The reaction product consists of 106 base pairs.

Table 4. 30

 Name

 Sequence Size 5´- 3´

 KIR2DS5-sense

 19pb SEQ ID NO: 1

 KIR2DS5-antisense

 23pb SEQ ID NO: 2

Table 5. Total alleles of KIR2DS5 detected that can be identified by the assay of the present invention

 2DS5 * 001

 2DS5 * 00502

 2DS5 * 0020101

 2DS5 * 006

 2DS5 * 0020102

 2DS5 * 007

 2DS5 * 0020103

 2DS5 * 00801

 2DS5 * 0020104

 2DS5 * 00802

 2DS5 * 00202

 2DS5 * 009

 2DS5 * 003

 2DS5 * 010

 2DS5 * 004

 2DS5 * 011

 2DS5 * 00501

 2DS5 * 012

 5

 10

Thus, a very preferable aspect of the invention is an oligonucleotide identified by a nucleic acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, and its complementary sequences. Another preferable aspect is an oligonucleotide with a sequence identity of at least 95%, 91%, 89% or 87% with SEQ ID NO: 1 or SEQ ID NO: 2. Another more preferable aspect is its use as a primer for sequence of the human genome, and another more preferable aspect is its use, or that of its functional variables, for the detection of the KIR2DS5 gene by the polymerase chain reaction.

Within the scope of the present invention, a nucleic acid sequence capable of specifically hybridizing with a DNA target sequence adjacent to or contained in the human KIR3DS5 gene is defined as a functional variable of a primer.

The position of de novo primers designed in the present invention is specific to the KIR2DS5 gene (Figure 2) and are capable of amplifying the corresponding region of exon 4, which encodes part of the extracellular domain D1 of the KIR2DS5 protein. No extra reactions have been detected for any other KIR gene.

The most preferable aspect of the present invention is a kit for carrying out the process of the invention, comprising specific reagents for the detection of the KIR2DS5 gene in a biological sample of a subject, instructions for the use of

said reagents and a device for determining whether the result of said detection is indicative of the genetic predisposition of said subject to develop non-viral liver cirrhosis from comparison with a control. Another very preferable aspect is a kit for determining the predisposition to develop non-viral alcoholic cirrhosis in a subject, comprising at least one of the primers identified as 5 SEQ ID NO: 1 and SEQ ID NO: 2, or its sequences complementary or its functional variables.

BRIEF DESCRIPTION OF THE FIGURES 10

Figure 1: Shows the frequency comparisons of the activating genes KIR2DS1, KIR3DS1 and KIR2DS5 observed in cirrhotic patients without viral infection (striped bars), with viral infection (mottled bars) and in healthy controls (white bars). An increased frequency of these genes was observed in patients with non-viral alcoholic cirrhosis. fifteen

Figure 2: Schematic representation of the genetic map of the KIR2DS5 gene showing the gene position of the different protein structural motifs it encodes. It is not drawn to scale, intronic areas have been reduced for better representation. OD, D1 and D2 represent the different extracellular domains 20 from more distal to more proximal positions of the plasma membrane, respectively. Cyt corresponds to the intracytoplasmic domain. The black dates mark the location of the sense and antisense primers of the invention in exon 4 corresponding to the extracellular domain (D1).

 25

Figure 3: Photograph of the result of a 3% agarose gel electrophoresis showing the result of the genotyping of the KIR2DS5 gene. For visualization, 5 microliters of sample were added per well, with a migration distance of approximately 3 cm, and stained with RedSafe (Intron biotechnology). The 106 base pair band corresponds to the amplification of the KIR2DS5 + gene in those 30 patients who present it (P1-4). M; Molecular weight marker (ΦX174 DNA-Hae III Digest (New Englands Biolabs). P1-4, KIR2DS5 + patients with non-viral alcoholic cirrhosis. C5-6, healthy controls KIR2DS5 + and p7-8 sick patients KIR2DS5-.

 35 EXAMPLES

With the intention of showing the present invention in an illustrative manner but in no way limiting, the following examples are provided.

Example 1: KIR gene profile of a healthy population or of patients. 5

To determine the prevalence of KIR genes, DNA samples extracted from anticoagulated peripheral blood were used, available at the Immunology Service of the Virgen de la Arrixaca Hospital in Murcia. The comparisons used data from the sick male population accompanied or not by viral infection, as well as from a healthy population. The presence of known KIR genes in the total of 10 samples (Table 1) was initially analyzed by PCR-SSO (“Sequence Specific Oligonucleotide”) techniques with Luminex technology (Tepnel Lifecodes) and PCR-SSP (“Single Specific Primer”) as described by Vilches (Vilches C et al. “Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments.” Tissue Antigens. 2007; 70 (5): 415-22). The specific presence of the KIR2DS5 gene, in the D1 domain, was detected with the following amplification and hybridization protocol. For each amplification reaction, 100 ng of genomic DNA contained in 10 µl of PCR buffer (67 mM Tris-HCl, pH 8.8, 16 mM (NH4) 2SO4, 2mM MgCl2, 0.01% Tween-20, 100 mM dNTPs) containing 0.3U of Taq polymerase (EcoTaq; Ecogen SRL, Barcelona, Spain). He underwent an initial denaturation for 2 20 min at 950C followed by 10 cycles of 10s at 940C plus 40s at 730C, in addition to 20 cycles of 20s at 940C, 20s at 680C and 30s at 720C (ABI2720 Thermocycler, Applied Biosystems, Foster City , CA). Subsequently, the amplification products were visualized by 3% agarose gel electrophoresis with 5 microliters of sample per well, with a migration distance of approximately 3 cm, and stained with 25 RedSafe (Intron biotechnology),

Example 2: Determination of KIR2DS5 in patients with alcohol consumption habits with symptoms of non-viral alcoholic cirrhosis.

Samples of male patients were considered according to the previous example, with 30 symptoms of cirrhotic disease in final staging and with alcohol consumption habits. The specific and selective amplification of the fragment specified in the invention of 106 bases of the KIR2DS5 gene in each of the samples was carried out. The result allowed us to observe a band, specific and indicative of the presence of the KIR2DS5 gene (Figure 3). 35

Example 3: Determination of KIR2DS5 in the genotype of a healthy patient with alcohol habits and without cirrhosis.

In samples of patients who presented alcohol habits but no symptoms of liver cirrhosis. The procedure of the previous example 5 was repeated to evaluate the presence or absence of KIR2DS5, not observing genetic amplification (Figure 3, see C5-C6)

10

Claims (15)

1. Procedure for the prediction of non-viral alcoholic liver cirrhosis in a subject, characterized in that it comprises the following stages: - detecting the presence of the KIR2DS5 gene in a biological sample obtained from said subject, - comparison of the result of the previous detection with a control, and - Evaluation of the genetic predisposition of said subject to develop non-viral liver cirrhosis from the previous comparison.  10 2. A method according to claim 1, characterized in that said biological sample is a blood sample. 3. A method according to claim 1, characterized in that said biological sample is a tissue sample. 4. A method according to claim 1, characterized in that said biological sample is a saliva sample. 5. A method according to any of claims 1 to 4, characterized in that said subject is male. A method according to any one of claims 1 to 5, characterized in that said detection comprises a polymerase chain reaction. twenty 7. Oligonucleotide characterized in that it is identified by a nucleic acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, and its complementary sequences. 8. An oligonucleotide according to claim 7, characterized in that it has a sequence identity of at least 95% with SEQ ID NO: 1 or SEQ ID NO: 2. 25 9. An oligonucleotide according to claim 8, characterized in that said identity is at least 91%. 10. An oligonucleotide according to claim 9, characterized in that said identity is at least 89%. 11. An oligonucleotide according to claim 10, characterized in that said identity is at least 87%. 12. Use of an oligonucleotide according to any of claims 7 to 11, as a primer for the human genome sequence. 13. Use of a primer according to claim 12, or its functional variables, for the detection of the KIR2DS5 gene by the polymerase chain reaction. 35 14. Kit for carrying out the process according to claims 1 to 6, characterized in that it comprises specific reagents for the detection of the KIR2DS5 gene in a biological sample of a subject, instructions for the use of said reagents and a device for determining whether the result of said detection is indicative of the genetic predisposition of said subject to develop non-viral liver cirrhosis from comparison with a control. 15. Kit for the prediction of non-viral liver cirrhosis of a subject, characterized in that it comprises at least one of the primers identified as SEQ ID NO: 1 and SEQ ID NO: 2, or its complementary sequences or its functional variables.  10