Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-27
2003-12-09
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06660477
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns a method for the determination of data for the preparation of the presymptomatic or prenatal diagnosis of phakomatosis, in particular of tumor suppressor gene diseases, in particular of neurofibromatosis (type 1, type 2). Such methods are useful for children of parents suffering from a hereditary disease or their grandchildren to increase the probability of early detection of a new occurrence of the hereditary disease, or (if possible) of prenatal evaluation.
BACKGROUND OF THE INVENTION
The current state of the art for the preparation of the corresponding diagnosis, for this purpose, uses a mutation analysis of the DNA section coding for the characterizing gene for the hereditary disease. These tumor suppressor gene diseases include the autosomal dominant inherited neurofibromatoses.
Neurofibromatosis occurs in two types, the peripheral type, called type 1, which represents approximately 85% of the cases, and the “central type,” called type 2, which represents approximately 15% of the cases. Type 1 occurs with an incidence of approximately 1:3000, whereas type 2 occurs with an incidence of approximately 1:35,000. For descriptions of the clinical picture, reference is made to appropriate specialized medical books.
The drawback of the mutation analysis is that it is very time consuming. For example, the neurofibromtosis type 1 gene on chromosome 17 (NF1 gene) has 60 exons. A complete analysis of this gene using the known mutation analysis takes more than four months. Although the neurofibromatosis type 2 gene (NF2 gene) located on chromosome 22 is smaller, having only 17 exons, a complete analysis still takes more than one month. In addition, one drawback of the known mutation analysis is that in high-risk individuals the diagnosis can only be considered to have been confirmed by molecular genetic means if a mutation is found in the afflicted individual.
SUMMARY OF THE INVENTION
A problem of the present invention is to improve a method of the type mentioned in the introduction. The problem is solved according to the invention by means of a method according to the claims.
An advantage of the method according to the invention, in particular, is that it can be carried out very quickly. Thus, in all cases, the method according to the invention can be carried out in less than 2 weeks; moreover, if the procedure is accelerated, it can be carried out in approximately two days. The rapidity of the method according to the invention is particularly important in prenatal diagnosis. Moreover, the method according to the invention is also considerably more cost effective because of its simplicity than the mutation analysis known from the state of the art.
The method according to the invention is particularly advantageous in cases where the known mutation analysis was unable to detect any mutation in individuals who were carriers of a mutation. The method according to the invention now offers the only possibility, in sporadic cases, of ruling out, or confirming, neurofibromatosis of type 1 or 2 on a molecular basis. In this context, the exclusion of neurofibromatosis is of particular importance because, statistically, it is possible to rule out the disease in approximately 50% of the high-risk individuals. As a result, the invention not only allows the elimination of the cost-intensive mutation analyses and examinations, it also makes it possible to prevent the anxiety an individual undergoing the examination may have concerning the possibility of having inherited the disease. In addition, expensive clinical examinations are also not necessary.
In a preferred embodiment, the markers are relatively short gene-flanking or intragenic DNA sections (to 300 bp). This offers the advantage that material that may be available, for example in the form of paraffin blocks prepared after surgical interventions on skin tumors in cases with neurofibromatosis, can be used, because it is possible to amplify short DNA sections from most of the available paraffin blocks. A special advantage can be seen in the fact that, particularly in the case of neurofibromatosis, the tumor material can easily be collected by external interventions.
In an additional preferred embodiment, at least four different markers are amplified. In this manner an improved data base which prevents possible detrimental misjudgments can be created for later diagnosis.
In an additional advantageous embodiment of the invention, the diagnosis of neurofibromatosis of type 1 is prepared. For this purpose, at least one polymorphous microsatellite marker from intron 27 of the NF1 gene. Furthermore, it is preferred to use at least one additional polymorphous microsatellite marker from intron 38 of the NF1 gene. Optimal results can be achieved when a total of three or four markers from the introns mentioned are used. This is advantageous because it has been shown that, in a predominant number of the high-risk patients examined, at least one of the markers mentioned is informative. A marker is informative for a given individual if the corresponding marker is present in polymorphous form and having two different lengths on both copies of the heritable material. The markers mentioned thus guarantee that there are two peaks in the graphic representation of the markers based on the difference in length.
As the preparative step for the diagnosis, the physician can compare the two peaks of the graphic representation of the markers from the blood of the afflicted individual, first with the result of the graphic plotting of the length of the DNA microsatellite markers from the tumor, in order to establish the presence of LOH (loss of heterozygosity=LOH). Here the invention includes the knowledge that the neurofibromas of the individuals from which the tumor material was removed present a 30% loss of heterozygosity, in the case of the neurofibromatosis type 1. In the case of tumors associated with neurofibromatosis type 2, the LOH rate is even higher. Thus, based on the fact that NF1 patients present many neurofibromas, the probability is very high that LOH occurs in any of the neurofibromas of the patient, and thus that it is also present and can be detected in the tumor material made available. The LOH can be recognized in the graphic representation of the markers because in the tumor material only one peak or one imbalance of the two peaks of the corresponding marker can be recognized. Both findings mean that the corresponding tumor has lost an allele. After the detection of LOH, the same marker from the blood of the high-risk person is then examined.
In another embodiment of the invention, steps c), e), g), and i) of claim 4, are repeated at least once. In this manner, a loss of an allele can again be verified or confirmed. Thus support for the first result can be obtained, if in the case of LOH the loss of an allele can be confirmed in at least one of the additional examinations.
In an additional preferred embodiment such an LOH is verified, if possible, in at least one additional tumor of the afflicted individual, that is the above-mentioned steps are carried out with at least one additional tumor of the afflicted individual, if the tumor is available. In this manner the reliability of the data obtained can be further increased. This is particularly advantageous in prenatal diagnosis.
An additional embodiment example of the invention is also carried out by steps b), d), f), h) and j) of claim 4 with the blood of the parent who is not affected, if the high-risk patient is a child of both parents. In this manner it becomes possible to determine alleles that are not affected. This also leads, on the one hand, to an increase in the reliability of the data obtained, and, on the other hand, in some cases, it is indispensable in the evaluation of the data obtained for diagnosis. As an example pertaining to this, it is mentioned that it is possible that the graphical representation of the alleles of the afflicted individual shows that he/she has alleles A and B.
In the graphical representation of the alleles
Fish & Neave
Gunnison Jane T.
Jenner Jesse J.
Ketter James
Sandals William
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