Patent Application: US-74498808-A

Abstract:
disclosed is an in vitro method for diagnosing susceptibility to post - traumatic scar tissue formation , wherein from a biological sample of a patient the nucleotide of the - 509 position of the tgf - β1 gene is determined and if said - 509 position contains exclusively c , thus it is the homozygotic wild type allele , then said patient is considered to be susceptible to post - traumatic scar tissue formation . the invention relates furthermore to diagnostic kits for the detection of susceptibility to post - traumatic scar tissue formation , preferably for the detection of susceptibility to tracheal stenosis from a biological sample .

Description:
in the detailed description below , we shall demonstrate several examples concerning the method according the present invention and concerning the diseases , which can be diagnosed by performing the method according to the present invention , etc . however it is obvious for a person skilled in the art that only certain embodiments of the present invention are described to assist an artisan . clearly we have no intention to limit the scope of the present invention with the described examples , they are only assisting in the use of the present invention . during the diagnostic procedure of the present invention to identify said polymorphism , pcr - rflp process can be preferably used , which essentially consists of the amplification of the gene region with the polymorphism to be examined ( pcr ), the digestion by a restriction endonuclease and the analysis of the resulted fragment ( rflp ). we have to emphasize that the procedure according to the present invention will not be limited to the above pcr - rflp process . the polymorphism can also be identified by other known methods , like those , which are based on the identification of the nucleic acid sequence or on hybridization and other processes based on the detection of improper base - pairing . this process can be for example a pcr process and the subsequent sequencing , or a pcr - reaction with an allele - specific taqman test . according to one of the preferred embodiments of the present invention , the genetic factor — firstly ever indicated for the susceptibility to the development of post - traumatic tracheal scar - tissue — can be determined in a modest molecular biology laboratory , equipped minimally with the following instruments : pcr device for the amplification of the gene fragment harboring said polymorphism , gel electrophoresis device for separation of the fragments on an agarose gel , and gel documentation system for the photography of the agarose gel , evaluation of results and data recording . one of the preferred embodiments of the present invention can be performed by the following way : genomic dna - isolation ( approx . 1 hour ) is performed from 2 - 3 ml venous blood taken from the patient before intubation ; amplification of the gene region harboring the polymorphism by pcr ( approx . 2 - 3 hours ) with the following primers : digestion of the amplified dna - fragment by the ddei restriction enzyme ( approx . 2 hours ), genotyping on 5 % agarose gel , separating the low molecular weight fragments with high efficiency ( approx . 2 hours ). 46 + 74 bp fragments : c / c — it indicates , that the given individual is wild type at the - 509 position of the tgf - β1 gene one 120 bp fragment : t / t — it indicates , that the given individual is a homozygote mutant at the - 509 position of the tgf - β1 gene 120 + 74 + 46 bp fragments : c / t — it indicates , that the given individual is a heterozygote at the - 509 position of the tgf - β1 gene . therefore it is obvious that genotyping of the - 509c / t tgf - β1 position of a patient can be done within one working day . in case an intubated patient harbors the c / c wild genotype , indicating susceptibility to tracheal stenosis , the physician has the possibility to make the proper decision by which the risk of stenosis formation can be reduced . the following example further illustrates the present invention but should not be construed as in any way limiting its scope . at the department of oto - rhino - laryngology and head - neck surgery in cooperation with the department of dermatology and allergology , ( both at the university of szeged , hungary ) we performed the following experiment : 66 patients were enrolled to the study from which 30 patients had no reaction concerning the development of the post - traumatic tracheal stenosis after the intensive care intubation ; while in case of 36 patients , the above mentioned pathologic condition developed . patients &# 39 ; data are summarized in table 1 . we took venous blood from the patients and purified genomic dna and compared the prevalence of the four tgf - β polymorphisms known in the literature in the two patient groups . to test the four polymorphisms we used a simple molecular biology method , which was the above mentioned pcr - rflp . in accordance with our experimental results , one of the tested polymorphisms — the - 509c / t — showed significant difference in the allele distribution between the two patient groups , namely in the group where despite the prolonged intubation the tracheal stenosis did not develop , the ratio of the heterozygotes ( c / t ) was much higher at said position , while in case of patients where tracheal stenosis formed as a result of the intubation , the ratio of the heterozygotes was much lower . the processing of the data ( table 2 )— calculation of the so called odds ratio ( or )— showed that the individuals being homozygote wild types at the given position had 4 . 5 - times higher chance regarding the development of the tracheal stenosis than the other individuals who were heterozygotes on this position . our test results did not include the homozygote mutant individuals because the literature data as well as our assay showed that this genotype is rare within the caucasian population . the above mentioned denotes that the genotyping of the tgf - β1 - 509c / t polymorphism of the patients treated by intubation can help in the to estimation of susceptibility to tracheal stenosis . our assumption is particularly supported by the fact that according to the internet databases as well as by our data , half of the caucasian population is homozygote wild type while the other half is heterozygote for the given allele . accordingly , half of the population has four - times higher chance to develop post - intubation tracheal stenosis due to the intensive therapy than the other half of the population . the presented results make it possible to perform an easy and simple diagnostic procedure , which can select the above mentioned risk group . our result raise the possibility , that the tgf - β1 gene and / or the tgf - β1 protein coded by it can be introduced in future as a therapy target in the treatment of scarry tracheal stenosis . 1 . sengupta p ., et al . endotracheal tube cuff pressure in three hospitals , and the volume required to produce an appropriate cuff pressure . bmc anesthesiol . 2004 ; 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