DE19738908A1 - Assessing genetic susceptibility to neuro-dermatitis and asthma - Google Patents

Abstract

Method for determining individual genetic influences on the effect of pollutants on neurodermatitis and/or asthma patients and/or for planning a therapy regime for such patients comprises: (a) determining the allelic variability in one or more polymorphic genes affecting the expression or activity of one or more enzymes responsible for detoxification and/or detecting the presence of such genes in a tissue and/or body fluid sample from the patient; (b) using the resulting genetic profile of the patient to determine the expected expression or activity of the individual enzyme variants and the expected enzymatic phenotype of the patient; and (c) using the resulting values to determine individual pollutant exposure limits and/or a suitable therapy regime for the patient.

Description

The present invention relates to a ver drive to determine individual genetic pre dispositions for pollutant influences and with the Planning a therapy concept for people affected by Neurodermatitis or asthma affected or at risk are. Such methods are in the area of order world medicine and occupational medicine and the associated who used diseases such as neurodermatitis.

The environment of humans is burdened by environmental chemicals (xenobiotics = foreign substances for the living organism) of natural and human origin, which by today's standards are incompatible with higher life and are therefore unphysiological. They cause the disorders in difficultly regulated physiological systems that are difficult or impossible to repair at all. These foreign substances enter the organism through the air, skin or food. Water-soluble substances are usually quickly excreted. On the other hand, water-insoluble substances accumulate in the body - often in the fatty tissue (bioaccumulation) if they are not metabolized promptly. For this reason, humans and higher organisms have a complex enzyme system through which water-insoluble substances are made available for excretion. This complex enzyme system mediates the following reactions in two phases:
In the so-called phase I reaction, the environmental toxins are first implemented by enzymes from the cytochrome system in reactive intermediates, which can be acutely more toxic than the pollutants themselves. B. bind to cellular components and cause damage. However, chronic damage can usually be traced back to the water-insoluble starting substances.

The phase II reak following phase I tion suspends the reactive intermediates separable end products.

It is known that individual individuals differed enzyme activities for phase I reactions and which have phase II reactions.

These enzyme activities are crucial for that different sensi in different individuals activity on xenobiotics and thus for the outbreak of diseases caused by the influence of environmental  micro-caused with, such as Eczema or asthma.

To the individual resilience with the environment to determine chemicals or in order to treat per to determine the right therapy will be followed the prior art a stress test leads over to the detoxification ability and there closed with enzyme activity of this individual can be. This is done according to the state of the art by administering a test substance that is caused by the un testering enzymes is broken down, and subsequent Determination of the clearance of the individual for this substance.

The disadvantage of this is that the determination of the enzyme activity only through an additional burden on the Person with a chemical can be identified. Such a method of determination is prohibited especially for children or people with reduced General condition or persons known of is that they are already heavily polluted. Therefore, this procedure may not work in every case be turned around, especially not with people which due to their pollution level the Measurement of the enzyme activity to determine the appropriate Therapy would be particularly important.

It is therefore an object of the present invention Procedure for determining individual predisposition for pollutant influences and / or for planning a Therapy concept for neurodermatitis or asthma persons at risk or affected to provide that avoids the above disadvantages and also for previously stressed or weakened people  or can be used on children.

This object is achieved by the method according to the invention ren according to the preamble of claim 1 in verbin solved with its characteristic features.

By the inventive method for determination individual genetic influences for pollutant actions and / or to plan a therapy con Zeptes for those at risk of neurodermatitis and / or asthma Allelic Variabi becomes the person or person who is ill lity or the absence or presence of a Gens determined based on expression or acti vity of one or more for the detoxification of the human body responsible enzymes ken. This means that the state form of each genes underlying the enzymes are determined and so the expected enzyme activity is determined becomes. This procedure does not immediately the enzyme activity as determined in the prior art, but independent of an activity determination of the Enzyme the expected genetically predetermined acti vity determined.

This method can therefore be used independently of the examining person in the laboratory and is due to the high degree of automation Genetic testing inexpensive and fast perform.

Advantageous further developments of the invention Procedures are be in the dependent claims wrote.

The expression or activity of an enzyme be  Influencing genes can be those for an inducer of Enzyme coding genes or that for the enzyme self-coding gene. Particularly meaningful Results are obtained by examining the Genes responsible for the activity of glutathione-S-trans are determining. The glutathione-S-transferase is one of the most important phase II enzymes and be therefore agrees very much about his activity Speed and thus the capacity of the detox process. The glutathione-S-transferase is in two ways through a genetic polymor excellent phism. For one, one or at de the (located on the paired chromosome) for the genes coding for glutathione-S-transferase GSTM1 is missing and therefore one if two are present Gene GSTM1 homozygous type, one in only one GSTM1 gene heterozygous type and one in the absence both genes, in turn, are homozygous. To the others lead the exchange of guanine for cytosine at position 2619 from an enzyme type B to an enzyme type A, the heterozygous AB type having the highest po has potential enzyme activity. By the determination measurement of an individual's genomic allele variations around the potential detoxification potential ability and thus the potential load limit values as well as the resulting ones for this individual determine appropriate therapy.

A particularly important enzyme of the phase I reaction way is the aryl hydrocarbon hydroxylase. The higher the activity of the enzyme is the more substrate can therefore be reactive intermediate per unit of time products are metabolized so that at a low activity of the phase II enzymes  Detoxification problem or a low resilience the person may experience pollutants.

Another particularly meaningful system is consequently the gene CYP1A1, which codes for the aryl hydrocarbon hydroxylase. On the one hand, an exchange of an adenine for guanine at position 4889 of exon 7 leads to a genetic polymorphism with this gene, which is associated with a changed phenotypic enzyme activity. On the other hand, an exchange of thymine for cytosine at position 264 downstream of the poly-A additional signal leads to a further genetic polymorphism of the aryl hydrocarbon hydroxylase. The enzyme that arises after replacing the thymine with cytosine is characterized by a particularly high inducibility.

The Ver according to the invention can be particularly advantageous run on a sample of whole blood or another Body fluid can be performed.

Evidence of allelic variability is given in otherwise known manner carried out in that the DNA isolated, duplicated and then is digested by suitable restriction enzymes. The generated fragments, the length of which Occurrence of the above point mutations in the depends on the respective genes, can subsequently by Separation for example by electrophoresis and staining exercise can be demonstrated.

For the duplication of the genes are particularly suitable especially the primers mentioned in claim 16.  

In the following some embodiments of the described method according to the invention. Show it:

Fig. 1, six suitable for determination of the glutathione S-transferase variants primer sequences;

Fig. 2 is suitable for determining the five aryl Hydrocarbon- hydroxylase variants primer sequences;

Figure 3A shows the relationship between the absence or presence of the gene and the GSTM1 genotype Phenom the enzyme activity of glutathione S-transferase.

Figure 3B is the classification of the enzyme activity of the Glu tathion-S-transferase according to the genotypes of GSTM1 gene.

Fig. 3C assessing the detoxification ability of the classification of the enzyme activity of the glutathione-S-transferase according to FIG. 3C;

4A is the relationship between the phenotype of the enzyme activity of the aryl Hydrocarbon- hydroxylase and the genotype of the CYP1A1 gene.

FIG. 4B, assessing the total enzyme activity according to the classification of Fig. 4a, and

Fig. 4C assessing the detoxification ability of the classification according to Fig. 4b.

The glutathione-S-transferase is one of the most important most enzymes of the phase II pathway, the intermediate human products from phase I (e.g. epoxies, Nitrosamines) to separable end products through Kon jugation with glutathione deactivated. The reduced one Form of glutathione has a nucleophilic 5H Group easily with compounds with electrophilic C atoms reacted to the thioether. The conjugates will not excreted uneconomically as such, but as "stripped-down" mercarpturic acids, the quan in the urine are detectable titatively. The amount depends Mercapturic acid depends on how well the enzyme Glu tathion-S-transferase glutathione on the substance can transmit. The highest possible activity of the Glutathione-S-Transferase speaks for an efficient Conjugation and rapid detoxification, a diminished Slow detox activity.

The human erythrocyte is 25% of the time no enzyme activity, medium in 50% of cases Enzyme activity and very high in 25% of cases Enzyme activity of glutathione-S-transferase. The Glutathione-S-transferase is in two ways genetically polymorphic. For one, one of the two human beings located on the paired chromosomes genes for GSTM1, which are responsible for glutathione S Transferase coded, missing, the other leads to Exchange of a guanine for a cytosine in position 2619 on two differently active enzyme forms A and B.  

To determine the individual resilience or to determine a suitable therapy is in ei The first step was to decide whether the gene in general is present or not. To avoid misinterpretation Avoid the absence of the reaction for the GSTM1 gene internal control of the reaction to the Pseudogen GSTM4 always present. Gets you just have a reaction to the control GSTM4 is the zero genotype, i. H. the missing of both genes for GSTM1. Otherwise, from before present at least one functional gene be gone.

Checking whether the gene is generally present is carried out in an otherwise known manner as follows leads.

The DNA is isolated from a blood sample using a commercially available known kit. A polymerase chain reaction is then carried out with the following specifications, the oligonucleotides shown in FIG. 1 being used as primers:
PCR premix: 93 µl consisting of:

8 µl MgCl ₂ (2 mM in the batch)
10 µl 10 x polymerase buffer
2 µl dNTP's (0.2 mM in the batch)
2 µl primer 1 (1 pmol in the batch)
1 µl primer 2 (1 pmol in the batch)
1 µl primer 3 (1 pmol in the batch)
69 µl dH ₂ O

Taq polymerase:
0.5 µl are mixed with 0.5 µl 1 x polymerase buffer per batch, a total of 1 µl pipetted (2.5 U in the batch).

6 µl of DNA and 93 µl of the PCR premix are placed in the PCR tubes. Then 1 ul of Taq polymerase mixture (1: 1) is added and the PCR cycle is started. The PCR cycle is carried out in the following phases:

The primer for internal control (primer 3) sets within the gene segment searched for and leads together with primer 1 to a product of 202 bp between positions 1156 and 1357 and must always be ampli be infected. The possibly deleted Genab cut for the GSTM1 gene follows Heading 1430. The GSTM1 gene is completely present a larger product of 275 bp between positions 1156 and 1430. Thereby ensures that no incorrect statement via the deletion and grouping to zero type is made.

The products are separated using gel electro phorese in 2% agarose at 100 V and the detection by coloring in an ethidium bromide bath. The gangs are visible under UV light and documented by Polaroid bar. To determine the size of the as a band too  a marker is added to the observing gene segment sets whose size is known.

If a functional GSTM1 gene is present, it can be present in variants A or B, which - as described below - is investigated by using specific primers and then digesting the restriction. The procedure is the same as for the control of the presence of the gene, so that in the fol lowing only the differing information is given, reference being made again to FIG. 1 with regard to the primers.

PCR premix: 93 µl consisting of:

8 µl MgCl ₂ (2 mM in the batch)
10 ul 10 × polymerase buffer
2 µl dNTP's (0.2 mM in the batch)
1 µl primer 4 (1 pmol in the batch)
1 µl primer 5 (1 pmol in the batch)
2 µl primer 6 (1 pmol in the batch)
69 µl dH ₂ O

Taq polymerase:
0.5 µl are mixed with 0.5 µl 1 x polymerase buffer per batch, a total of 1 µl, pipetted (2.5 U in the batch).

6 µl of DNA and 93 µl of the PCR premix are placed in the PCR tubes. Then 1 ul of Taq polymerase mixture (1: 1) is added and the PCR cycle is started. The PCR cycle has the following phases:

Primers 4 and 5 amplify DNA with cytosine and DNA, respectively with guanine, so that with the counter primer 6 a 132 Ba Pair long fragment of the GSTM1 gene for all cases le is reproduced.

The fragments are then differentiated with respect to the guanine / cytosine following the PCR by restriction digestion with the endonuclease HaeII. This endonuclease can only cut the gene GSTM1 if there is a cytosine instead of a guanine at position 2619 which, after expression of the gene, leads to the incorporation of an asparagine in place of lysine at position 172 of the glutathione-S-transferase. The endonuclease HaeII then cuts between the cytosine and an adjacent pyrimidine base. Restriction digestion with HaeII (20 U / µl) is carried out using the following approach:

15 µl PCR mix
2.5 µl 10 X reaction buffer
2.5 µl BSA (1:10)
5 µl enzyme (0.5 µl HaeII 10 U / µl plus 4.5 µl 1 x reaction buffer)
Incubate at 37 ° C for 2 h.

Genotype B remains completely uncut, so that only fragments with a size of 131 bp occur during genotype A by the endonuclease HaeII is cut so that fragments with a  Size of 113 bp and 119 bp occur. The method cannot distinguish whether the genotype A0 or AA or whether the genotype is B0 or BB. The heterozygous mixed type AB shows both uncut as well as cut fragments of all Sizes of 3% by electrophoresis Agarose for small fragments separated at 100 V and by coloring in the ethidium bromide bath and under UV light be made visible.

Figs. 3A to 3C show the analysis of the obtained here by knowledge of the genetic refund from an individual.

Fig. 3A shows the assignment of the basic genetic constellations of the two human GSTM1 genes to the expected activity of glutathione-S-transferase. The density of the hatching corresponds to the expected enzyme activity.

In the absence of both genes on the two chromosomes no enzyme activity expected. If available only a genotype, for example by genotype A0 or AA or B0 or BB is the expected enzyme assess activity as a means. If the heterozygous genetic type AB can be high Expect enzyme activity of glutathione-S-transferase.

Fig. 3B shows the division of enzyme activity into three different levels depending on the type of Ge. It also shows here that the highest enzyme activity in the heterozygous genotype AB, since no enzyme activity is to be expected in the absence of both GSTM1 genes, that is to say in genotype 00.

FIG. 3C shows the assignment of the detoxification ability to these enzyme activity levels determined in FIG. 3B. If the high enzyme activity of the genotype AB is expected, the detoxification ability is classified as sufficient. In the A0 / AA / B0 / BB genotypes, the mean enzyme activity is correlated with a mean detoxification ability. If there is no enzyme activity due to genotype 00, the ability to detoxify is greatly reduced due to the lack of expression of glutathione-S-transferase. For people with genotype 00, the usual pollutant limits have to be reconsidered. In the same way, the required therapy of people suffering from environmental diseases or people at risk can be concluded and these can be adapted to the individual's detoxification ability and to the individual exposure limit values.

The aryl hydrocarbon hydroxylase also has one genetic polymorphism. That for this enzyme coding gene CYP1A1 is inducible enzyme and its activity poly morph.

There are three different types of inducibility ne genotypes A, B and C, which predominate the homozygous type A the interface for the endo nuclease MSPI is missing. Through a one-base exchange from thymine to cytosine at position 264 upward from the poly-A additional signal becomes a bay generated the restriction enzyme MSPI, only between two cytosi next to each other can cut. The homozygous genotype for this rare allele is denoted by C, while he terozygote genotype is designated B.  

The detection of this polymorphism is carried out in an otherwise known manner by isolating the DNA from a whole blood sample of a person and then amplifying the DNA by a polymerase chain reaction. The polymerase chain reaction is carried out under the following conditions, the primers mentioned being shown in FIG. 2:

PCR premix: 93 µl consisting of:

8 µl MgCl ₂ (2 mM in the batch)
10 µl 10 x polymerase buffer
2 µl dNTP'2 (0.2 mM in the batch)
2 µl primer 1 (2 pmol in the batch)
1 µl primer 2 (1 pmol in the batch)
70 µl dH ₂ O

Taq polymerase:
0.5 µl are mixed with 0.5 µl 1 x polymerase buffer per batch, a total of 1 µl pipetted (2.5 U in the batch).

6 µl of DNA and 93 µl of the PCR premix are placed in the PCR tubes. Then 1 µl Taq polymerase mixture (1: 1) is added and the PCR cycle is started. The PCR cycle has the following phases:

The reaction products are 335 bp.

Following the polymerase chain reaction (PCR), a restriction digest is carried out with the endonuclease MSPI. Restriction digestion conditions are as follows:
Restriction digestion with MSPI (20 U / µl):

15 µl PCR mix
2.5 µl 10 X reaction buffer
2.5 µl dH ₂ O
5 µl enzyme (1 µl MSPI 20 U / µl plus 4 µl 1 x reaction buffer)
Incubation at 37 ° C overnight.

The common homozygous genotype A remains uncut and therefore only has fragments of 335 bp in size, the rare genotype C has fragments of 206 bp and 129 bp in size and is completely cut, d. H. it no fragments of 335 bp in size can be observed respect, think highly of. The heterozygous mixed type B has both cut and cut fragments of all three Sizes. The fragments are analyzed using gel electrophoresis se in 3% agarose for small fragments at 100 V. separated and by staining in the ethidium bromide bath and made visible under UV light. Every gel becomes one Markers added to the size of the reaction products to verify, and can doku through Polaroid photos be mented.

The second polymorphism of the CYP1A1 gene, the one Effect on the activity of aryl hydrocarbon Hydroxylase has an exchange of adenine against guanine at position 4889 in exon 7 of the CYP1A1 gene that is to be incorporated after expression of valine instead of isoleucine in the aryl hydro carbon hydroxylase at position 462 of the HR2 region leads.  

To detect this polymorphism, DNA is isolated from a whole blood sample in an otherwise known manner and a duplication of this DNA is carried out by a polymerase chain reaction under the conditions specified below, again the primers mentioned here being shown in FIG. 2.

PCR premix: 93 µl consisting of:

8 µl MgCl ₂ (2 mM in the batch)
10 µl 10 x polymerase buffer
2 µl dNTP's (0.2 nM at the start)
1 µl primer 3 or 4 (1 pmol in the batch)
1 µl primer 5 (1 pmol in the batch)
71 µl dH ₂ O

Taq polymerase:
0.5 µl of 0.5 1 x polymerase buffer are added per batch, a total of 1 µl pipetted (2.5 U in the batch).

Two approaches are carried out in parallel for each examination set, once with primers 3/5, the other time with primers 4/5 to detect the point mutation animals. Primers 3 and 4 end up with the Adenine / Guanine distinction. The high binding spec fity enables it to be parallel in the two approaches to assess whether only one of the binds both primers or both.

There are 6 µl of DNA and 93 µl of the PCR premix presented in the PCR tubes. Then 1 µl Taq polymerase mixture (1: 1) added and the Ther mo cycle started.  

The thermal cycle has the following phases:

The reaction products are 209 bp in size and form the homozygous isoleucine type only in the approach which contains primers 3 and 5. In the heterozygous Isoleucine / valine mixed type can be found in both PCR Approaches reaction products in the homozygous rare Valine / valine type only with primer 4 and 5.

The fragments are separated using Gelelek trophoresis in 3% agarose at 100 V and the night the individual bands of the fragments by staining in the ethidium bromide bath. The bands are under UV light visible and documentable via Polaroid. Any gel a marker is added to determine the size of the reak reliable product verification.

FIGS. 4A to 4C show the determination of the individual expected activity of aryl hydrocarbon hydroxylase from - as mentioned above - ge wonnenen results via the genetic Polymor homomorphism of the aryl hydrocarbon hydroxylase.

Fig. 4A shows the relationship between the waiting to he activity of aryl hydrocarbon hydroxylase with the occurrence of the genotypes A, B or C, as well as the occurrence of the genotypes Ile / Ile, Ile / Val or Val / Val. The stronger hatching means a higher enzyme activity. This division into four different levels of enzyme activity is shown again in FIG. 4B.

Fig. 4C shows the relationship between the potential expected individual detoxification ability and the expected activity of the aryl hydrocarbon hydroxylase of the individual. A high enzyme activity, as can occur due to a high inducibility (depending on the genotypes A, B, C) or due to a high activity of the individual enzyme (depending on the genetic makeup with regard to the Ile / Val polymorphism) due to the intensified phase I reaction to an increased formation of reactive intermediates and therefore to an acute higher pollution. If the overall activity of the aryl hydrocarbon hydroxylase is low, the phase I reaction is less pronounced, so that the acutely dangerous intermediates are obtained in a smaller amount.

From these values with regard to the expected indi vidual detoxification ability can now be reduced to a ge appropriate therapy of the respective person closed become.

By combining the analyzes of the genetic Aryl hydrocarbon polymorphisms Hydroxylase and the glutathione-S-transferase, such as Described above, a further improved Ge total statement about the expected load limit values with pollutants, detoxification ability or suitable therapy for chronic environmental cranes sufferers or people at risk be made. For example, high assets aryl hydrocarbon hydroxylase in connection  with a low activity of glutathione-S-trans ferase to a particularly strong enrichment of the reactive intermediates of the phase I reaction, wuh rend when there is a high or very high share vity of glutathione-S-transferase from a contemporary hen breakdown of the intermediate products can be assumed can, even if the activity of the aryl hydrocarbon Hydroxylase is increased. It is understood that in different therapies or a The therapy of different dimensions are indicated.  

SEQUENCE LOG

Claims (16)

1. A method for determining individual genetic influences for pollutant effects and / or planning a therapy concept for people at risk or suffering from neurodermitis and / or asthma, characterized in that the allelic variability of one or more polymorphic genes, which affect the expression or Activity of one or more enzymes responsible for detoxification of the human body, and / or their presence in a tissue and / or body fluid sample of the person recorded and the expression or activity of the person to be expected from the genetic makeup of the person recorded in this way individual enzyme variants and the expected enzymatic phenotype of the person are determined and from these values individual exposure limit values and / or a therapy concept suitable for this person is determined. 2. The method according to claim 1, characterized in that for an induc gene coding for the enzyme and / or the gene for the gene encoding the enzyme are examined.   3. Proceed according to at least one of the preceding claims 1 and 2, characterized in that the the transcript tion and activity of the enzymes glutathione-S- Transferase and / or aryl hydrocarbon hydroxyla influencing allelic variability of the Gene GSTM1 or CAP1A1 is determined. 4. The method according to claim 3, characterized in that the absence of a or both of the two human GSTM1 genes is determined. 5. The method according to claim 3 or 4, characterized in that the GSTM1 gene an exchange of a guanine for expression of a type B enzyme leads to cytosine Position 2619, which is used to express an enzyme type A leads, is examined. 6. The method according to at least one of claims 3 to 5, characterized in that the detoxification ability of the person according to the sequence of the Genoty pen
AB <AA = A0 = BB = B0 <00
is determined in decreasing order, where A and B stand for genes which encode an enzyme of type A or type B and 0 means the absence of a GSTM1 gene. 7. The method according to at least one of claims 3 to 6, characterized in that the coding for the aryl hydrocarbon hydroxylase gene CYP1A1 on an exchange of an adenine, which leads to the incorporation of an isoleucine in the enzyme, against guanine, which leads to the incorporation a valine instead of the isoleucine leads into the enzyme, is examined at position 4889 of exon 7 of the CYP1A1 gene. 8. The method according to claim 7, characterized in that the detoxification ability of the person according to the sequence of genetic equipment
Ile / Ile <Ile / Val <Val / Val
is determined in decreasing order. 9. The method according to any one of claims 3 to 8, characterized in that the CYP1A1 gene an exchange of thymine for cytosine Position 264 downstream of the poly A addition nal signal is examined.   10. The method according to claim 9, characterized in that the detoxification ability according to the sequence of the genetic equipment
Thymine / thymine <thymine / cytosine <cytosine / cytosine
can be determined in decreasing order. 11. Proceed according to at least one of the preceding claims 1 to 10, characterized in that the determination at a whole blood test. 12. Proceed according to at least one of the preceding claims 1 to 11, characterized in that from the sample DNS isolated, duplicated and then in traditionally the allelic variability is determined. 13. The method according to claim 12, characterized in that the DNA by Poly reproduced merase chain reaction (PCR), digested by suitable restriction enzymes and then the generated DNA fragments be separated and colored. 14. The method according to claim 13, characterized in that as restrictions zyme endonucleases can be used. 15. The method according to any one of claims 13 or 14, characterized in that the coloring with Ethidium bromide is made and the DNA fragments be separated by gel electrophoresis.   16. The method according to at least one of claims 13 to 15, characterized in that the following oligonucleotides are used as primers for the polymerase chain reaction for the detection of the genetic polymorphism of glutathione-S-transferase:
and for the detection of the genetic polymorphism of aryl hydrocarbon hydroxylase: