Marker sequences for diagnosing, and for controlling the therapy of patients with tesosterone deficiency

Abstract

The present invention relates to a method for the diagnosis, prognosis, risk stratification and therapy control of the lack in testosterone or testosterone deficiency in patients and patient populations, and to the use of suitable marker sequences and to a test kit.

Description

[0001] Marker sequences for the diagnosis and therapy control of patients with testosterone deficiency

[0002] The present invention relates to a method for the diagnosis, prognosis, risk stratification and therapy control of testosterone deficiency or testosterone deficit in patients and patient populations as well as the use of suitable marker sequences and a test kit.

[0003] Testosterone (17ß-hydroxyandrost-4-en-3-one) is a male sex hormone, or androgen, produced in the testes (gonads). It is known to promote male physical characteristics, such as muscle mass and body hair, as well as increase libido and support sperm production.

[0004] Testosterone deficiency, also known as hypogonadism, refers to a condition in which the testes do not produce enough testosterone or in which the body cannot properly utilize the testosterone produced due to age, injury, or illness. Genetic causes may also be present.

[0005] A testosterone deficiency can have many effects on the body, including reduced muscle mass and strength, increased body fat, decreased bone density, reduced libido, and erectile dysfunction. It can also lead to fatigue, depression, mood swings, and decreased mental acuity.

[0006] The diagnosis of testosterone deficiency is made through blood tests to measure testosterone levels in the body. If a deficiency is diagnosed, it can be treated with hormone replacement therapies, such as the administration of testosterone injections, gels, or patches. Epigenetics refers to changes in

[0007] Gene expression that is not due to changes in the DNA sequence itself. Instead, epigenetic mechanisms, such as chromatin structure and DNA methylation, regulate which genes are active or inactive and thus which proteins are produced.

[0008] Chromatin is the structure in which DNA is packaged within the cell nucleus, and DNA methylation is a process by which methyl groups are attached to the DNA, altering the chromatin structure. These epigenetic changes can be permanent or temporary and are influenced by various factors such as age, environment, and lifestyle.

[0009] Epigenetic changes can have important effects on health and development. For example, an aberration in the methylation of certain genes can increase the risk of certain diseases or even influence the course of the disease. An aberration in chromatin structure can also lead to the lack of expression of certain genes, which in turn can lead to a developmental abnormality.

[0010] Epigenetics also has the potential to contribute to the development of therapies by enabling the activation or deactivation of specific genes to treat specific diseases. Epigenetics is also being intensively researched to learn more about the underlying mechanisms of gene expression and to deepen the understanding of developmental processes and diseases.

[0011] CpG sites are regions of DNA consisting of a cytosine (C) and a guanine (G) linked by a phosphate bond. These CpG dinucleotides are not evenly distributed in DNA, but occur in groups called CpG islands. CpG sites play an important role in epigenetics because they are the primary target region for DNA methylation reactions. During DNA methylation, a methyl group is attached to the carbon atom of the cytosine, resulting in the formation of 5-methylcytosine (5mC). Methylation of CpG sites can cause gene expression in the surrounding region of DNA to be repressed, resulting in a reduction or even silencing of gene expression.

[0012] In fact, CpG islands are frequently found in promoter regions of genes involved in the regulation of gene expression. An aberration in the methylation of CpG islands can affect gene expression and contribute to various diseases or conditions, resulting in methylation or demethylation of the CpG sites or CpG islands. The most common form is hypermethylation of the CpG sites or CpG islands. Hypomethylation has also been described.

[0013] The identification of CpG sites or CpG islands is therefore of great importance in epigenetic research, particularly in the identification of epigenetic markers and their association with certain diseases.

[0014] Surprisingly, the inventors were able to identify epigenetic markers for the state of testosterone deficiency in a patient, particularly in relation to aging.

[0015] Therefore , an object of the present invention is to improve the therapy , prophylaxis and prevention of testosterone deficiency by means of drug treatment as well as with regard to clinical decisions .

[0016] A further object of the invention is also the therapy control by means of a prognosis or diagnosis of testosterone deficiency or testosterone deficiency patients. In particular, such testosterone deficiency patients who have not previously received any drug treatment, or preferably for the early detection of such testosterone deficiency patients, in particular for the prophylaxis or prevention of testosterone deficiency or testosterone deficiency.

[0017] These problems are solved by the technical teaching of the patent claims.

[0018] The object is achieved according to the invention by providing a method for the diagnosis or prognosis of patients with testosterone deficiency, comprising analysis of the methylation status of at least one CpG island of a marker sequence (MS) selected from MS No. 1 - 23 of a patient sample, wherein at least one modification by methylation and / or demethylation is indicative in comparison with the respective MS No. 1 - 23.

[0019] In a further preferred embodiment, the invention relates to a method for the diagnosis or prognosis of patients with testosterone deficiency, comprising analysis of the methylation status of at least one CpG island of a marker sequence selected from MS No. 1 - 23, wherein hypermethylation or hypomethylation of the respective MS No. 1 - 23 is present and indicative.

[0020] The results of the examples show that the marker sequences according to the invention are particularly suitable for patients aged 25-60 years, especially 30-55 years, see Figures 1 and 2.

[0021] In a further preferred embodiment, the invention therefore relates to a method for diagnosing or prognosticating patients with testosterone deficiency, wherein the patients are selected from the group 30-55 years of age.

[0022] The invention also relates to the use of one or more marker sequences according to the invention for stratifying and controlling therapy in patients with testosterone deficiency or testosterone deficiency. A further embodiment relates to the use according to the invention, characterized in that 2 or 3, preferably 4 or 5, particularly preferably 6, 7 or 8 or more different marker sequences, for example 10 to 20 different marker sequences, are used. Such a set of marker sequences is called a panel.

[0023] The invention also relates to a method for stratification, in particular for risk stratification, or for therapy control of a patient with testosterone deficiency or testosterone deficiency, wherein at least one marker sequence according to the invention is used to examine a sample from the patient.

[0024] The method according to the invention is particularly suitable for the early diagnosis or early detection of testosterone deficiency, in particular where no, i.e. asymptomatic, or few symptoms (supra) are detectable in the patient.

[0025] One embodiment relates to a method according to the invention for diagnosing testosterone deficiency, wherein the stratification or therapy control comprises decisions on the treatment and therapy of the patient, in particular the use, effect and / or dosage of one or more drugs, in particular testosterone or a testosterone derivative, a therapeutic measure or the monitoring of a disease progression as well as the course of therapy, etiology or classification of a disease, if applicable including prognosis.

[0026] Therefore, the invention relates to the use of marker sequences for stratification or therapy control of testosterone deficiency, wherein at least one marker sequence selected from the group of marker sequences MS No. 1-23, partial sequences or fragments thereof, homologs of the sequences MS No. 1-23 with at least 90% homology to a patient to be examined is determined, wherein at least one modification by methylation and / or demethylation is indicative in comparison with the respective MS No. 1-23.

[0027] A further embodiment of the invention relates to the use of the marker sequence(s) according to the invention for stratification or therapy control of testosterone deficiency, wherein the determination is carried out by means of an in vitro diagnosis.

[0028] In a preferred embodiment, the determination of the marker sequences takes place outside the human body and the determination is carried out in an ex vivo / in vitro diagnosis.

[0029] The marker sequences according to the invention could be identified by differential screening of samples from healthy volunteers with patient samples with testosterone deficiency, see examples.

[0030] Preferably, the analysis is carried out by amplification using a polymerase enzyme, a PCR or a chemical amplification reaction or other amplification methods, ie within the framework of MSP, HeavyMethyl, Scorpion, MS-SNUPE, MethylLight, bisulfite sequencing, methyl-specific restriction assays and / or digital PCR (see, for example, Kristensen and Hansen PCR-Based Methods for Detecting SingleLocus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment Clinical Chemistry 55:8 1471-1483 (2009) ).

[0031] To analyze the bisulfite convertibility of CpG islands, any known method for analyzing DNA methylation can be used. In a preferred embodiment of the method according to the present invention, the analysis of the methylation status comprises a method selected from methylation-specific enzymatic digestion, bisulfite sequencing, an analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, Ms-SNuPE, or other methods based on the detection of amplified DNA. These methods are well known to those skilled in the art and can be found in the relevant literature.

[0032] In a preferred embodiment of the method according to the present invention, the method is suitable for routine applications, for example, on a DNA array. Based on the above information and the relevant literature, one skilled in the art can adapt the above method to these settings as described above.

[0033] The term "testosterone deficiency" is defined according to the invention as the patient having less than 30 pg / ml or 40 pg / ml of free testosterone in the saliva in the age group 30-65 years. The diagnosis and prognosis are also to be understood in such a way that the test subject may be affected by testosterone deficiency in the future, even if he has normal testosterone values ​​at the time of sample collection, namely 30-140 pg / ml of free testosterone in the saliva.

[0034] "Diagnosis" or "prognosis" within the meaning of this invention means the positive determination or prediction or probability of the occurrence of testosterone deficiency or testosterone deficit using the marker sequences according to the invention as well as the assignment of the patient to the indication of testosterone deficiency or testosterone deficit. The term diagnosis encompasses medical diagnostics and related examinations, in particular in vitro diagnostics and laboratory diagnostics.

[0035] Furthermore, the invention relates to a method for

[0036] Stratify, particularly for risk stratification and / or therapy management of a patient with testosterone deficiency or testosterone deficit, for example in a patient with a very early stage of testosterone deficiency or

[0037] Testosterone deficiency .

[0038] "Stratification or therapy control" in the sense of this invention means that the method according to the invention allows decisions regarding the treatment and therapy of the patient, be it hospitalization of the patient, use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a disease progression as well as the course of therapy or etiology or classification of testosterone deficiency or testosterone deficit.

[0039] In a further embodiment of the invention, the term "stratification" particularly encompasses risk stratification with the prognosis of an "outcome" of an adverse health event.

[0040] In the context of this invention, "patient" is understood to mean any test subject - human or mammal, preferably male - with the proviso that the test subject is being examined for testosterone deficiency or testosterone deficit, in particular a therapeutic success or therapeutic improvement, or with the proviso that the test subject or individual receives or is to receive medication for the treatment of testosterone deficiency or testosterone deficit, such as by means of hormone replacement therapy.

[0041] According to the invention, the sequences to be examined in a sample are components of a body fluid, in particular blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, synovial fluid or a tissue extract from the patient.

[0042] However, sampling using a cheek swab is particularly preferred. The marker sequences MS Nos. 1-23 according to the invention are shown in Table 1 and can be clearly identified using the respective cited database entry (Internet: www . http : / / www . ewascatalog . org / ) (see cg and gene names including position / loci).

[0043] Table 1: Marker sequences (MS) from Homo sapiens

[0044] A further embodiment of the invention encompasses homologs of the marker sequences according to the invention. In particular, those homologs that exhibit 90%, 91%, 92%, 93%, 94%, or 95% identity, in particular 96%, 97%, 98%, 99% or more identity with the marker sequences according to the invention and are suitable for the use according to the invention—the detection of testosterone deficiency or testosterone deficiency (so-called "homologues," homologous marker sequences).

[0045] Partial sequences or fragments are sequences that are 50 to

[0046] 100 nucleotides, preferably 100-500, particularly preferably 500 to 1000 nucleotides of one of the marker sequences MS No. 1-23, such as oligonucleotides.

[0047] Such marker sequences of the respective genomic DNA for MS Nos. 1-23 are indicated in the sequences SEQ ID Nos. 1-12 according to Table 1 and should be read accordingly. SEQ ID Nos. 1-12 are included in the respective corresponding MS Nos.

[0048] A further preferred aspect of the present invention relates to a (test) kit for carrying out the method according to the invention, in particular based on the analysis of the bisulfite accessibility of CpG islands of the marker sequences MS No. 1-23, comprising components for carrying out the method according to the invention, in particular optionally comprising a bisulfite reagent and materials for analyzing the methylation status of the CpG islands selected from at least one marker sequence, in particular an oligomer.

[0049] The invention will now be described in more detail in the following examples and with reference to the attached figures and the sequence listing, without being limited thereto.

[0050] Examples and figures:

[0051] The subject's DNA is taken using the Oracollect sampling system (OraCollect 100 from Genotek) by taking a swab of the epithelial cells from the inner cheek according to the manufacturer's instructions.

[0052] The DNA is purified for analysis using the Qiagen DNA purification kit (QIAamp UCP DNA Micro Kit) according to the instructions.

[0053] The epigenetic analysis is performed on the Illumina Infinium R

[0054] MethylationEPIC BeadChip system according to the

[0055] Manufacturer information: Illumina DNA Prep Reference Guide (1000000025416 vlO); https : / / support . Illumina. com / documentation. html

[0056] Infinium MethylationEPIC v2.0 BeadChip, PDF https : / / support . Illumina. com / documentation. html

[0057] Noble AJ, Pearson JF, Boden JM, Horwood LJ, Gemmell NJ, Kennedy MA, Osborne AJ. A validation of Illumina EPIC array system with bisulfite-based amplicon sequencing. Peer J. 2021 Feb 10;9:el0762. doi: 10.7717 / peerj.10762. PMID: 33614276; PMCID: PMC7881719.

[0058] Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, Shen R, Gunderson KL. Genome-wide DNA methylation profiling using Infinium® assay. Epigenomics. 2009; 1(1):177-200. doi: 10.2217 / epi .09.14. -

[0059] Data Analysis

[0060] First, methylation levels were compared against internal controls (e.g., standards from an epigenetic database). In a second step, differentiation was performed against the control group of "testosterone supplementers" (these are subjects who have been taking testosterone for years).

[0061] The resulting iDat files were analyzed in R for statistical data analysis. Result files in idat format are read directly with R.

[0062] P package provided by MIT: https : / / web .mit.edu / ~r / current / arch / 1386_linux26 / lib / R / library / limma / html / read . idat . html

[0063] Heatmap analysis and display with the package in R

[0064] (The R Graph Gallery, https: / / r-graph-gallery.com > heatmap) Data is normalized chip by chip using internal controls.

[0065] The methylation intensity of the marker sequences mentioned is compared after offsetting against the internal standards (normalization).

[0066] Heat map analysis shows that individual methylation sites clearly indicate testosterone deficiency. A single methylation site can be sufficient for clinical, diagnostic, or prognostic information.

[0067] Methylation intensities were controlled against two different groups.

[0068] 1. Control group with normal testosterone levels. These data support the statement regarding the age dependence of testosterone levels.

[0069] 2. Control group with testosterone supplementation. This group indicates the markers necessary for prognostic or diagnostic purposes.

[0070] In order to differentiate between classic age-related testosterone deficiency and age-independent testosterone deficiency, the analysis was carried out comparatively against the control group (“healthy men of different ages”).

[0071] Figures 1 and 2 show the heat map of 184 investigated GpC islands depending on age and population groups ( low level testosterone group and control group ) .

[0072] Evaluation :

[0073] The comparison was made against the control group (healthy men with typical age-appropriate testosterone levels) and against the hormone supplementation group (reference + hormones). Identification of the relevant CpGs.

[0074] CpGs with a selected difference in relative methylation intensity greater than 1.1 (maximum on the scale = 2) between the "testosterone-deficient (low-level testosterone)" and the "supplementation" group were identified. The selected difference may also be smaller.

[0075] In addition, the dependence on age was examined.

[0076] For statistical validation, a significance test was performed for all relevant CpGs using a t-test, with p < 0.01 or less in each case.

Claims

Patent claims:

1. A method for the diagnosis or prognosis or risk stratification of patients with testosterone deficiency, comprising analysis of the methylation status of at least one CpG island of a marker sequence selected from MS No. 1 - 23 of a patient sample, wherein at least one modification by methylation and / or demethylation is indicative in comparison with the respective MS No. 1 - 23.

2. A method for the diagnosis or prognosis or risk stratification of patients with testosterone deficiency according to claim 1, wherein hypermethylation or hypomethylation of the respective MS Nos. 1 - 23 is present.

3. A method for the diagnosis or prognosis or risk stratification of patients with testosterone deficiency according to any one of the preceding claims, wherein the at least one CpG island of a marker sequence is selected from MS No. 1-12 with SEQ ID No. 1-12 and MS 13-23 with the said cg number or gene loci according to Table 1.

4. A method for diagnosing or prognosticating or risk stratifying patients with testosterone deficiency according to any one of the preceding claims, wherein the patients are selected from the group 30-55 years.

5. A method for diagnosing or prognosticating or risk stratifying patients with testosterone deficiency according to any one of the preceding claims, wherein a Early diagnosis or early detection of testosterone deficiency occurs.

6. A method for the diagnosis or prognosis or risk stratification of patients with testosterone deficiency according to one of the preceding claims, wherein therapy control of testosterone deficiency takes place, in particular decisions on the treatment and therapy of the patient, in particular use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a disease course as well as the course of therapy, etiology or classification of a disease. 7 . Kit for carrying out a method according to one of the preceding claims, comprising materials for analyzing the methylation status of the CpG islands selected from at least one marker sequence MS No. 1-23, in particular an oligomer thereof. 8 . Use of a kit according to claim 7 for carrying out a method according to one of claims 1 to 6 .

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