DE19910912C2 - New sequence variants of the human RET protooncogene and their use - Google Patents

Description

The invention relates to new sequence variants of the human RET protooncogene and their Use for the predictive diagnosis of congenital diseases, their cause either in a maturation disorder or in an uncontrolled cell growth in tissues have neurogenic origin.

The human RET protooncogene was cloned and first described in 1988 The locus is 10q11.2. A membrane-bound receptor hyrosine kinase was used as the gene product characterized that has a meaning for both embryonic migration and Differentiation of cells derived from the neural crest as well as for their regulation of the Cell proliferation. Diseases that result from a disruption of this process summarized under the term neurocristopathies. In addition to multiple endocrine Type 2 neoplasia syndromes (MEN2) and familial medullary thyroid carcinoma (FMTC) are also the neuroblastomas, ganglioneuromas and the disease Hirschsprung and Undine syndrome belonged to this group. It is assumed, that the neoplastic diseases of this type of form an unspecific activation and reduced activity of the receptor tyrosine kinase due to the dysontogenetic disorders Is basically.

The invention has the aim of variants, polymorphisms, mutations and resulting Haplotypes in the DNA sequence of the germline of the human RET protooncogene determine and determine their correlations with disease dispositions. Starting from A method for molecular genetic diagnosis of these correlations is intended Disease disposition as well as to predict the severity and course of the disease be developed. The aim is to establish a genetic inheritance model in which Follow a prophylactic therapy that is both surgical and viable can have a medicinal character. The object is achieved according to the claims that Subclaims are preferred variants.

It was found that in addition to 4 already known polymorphisms (at positions 135, 2071, 2307 and 2710) and 2 known mutations (at positions 2372 and 2690) of the coding region of the sequence of the human RET protooncogene both in the coding as well as the non-coding region further variants are available. It  it was also found that these genetic variants with the disposition for different Diseases, e.g. B. correlate the Hirschsprung disease.

The invention then relates to the sequence of the human RET protooncogene, which is located at positions 135, Intron2 ds + 9, 447, 785, 799, 885, 972, 1013, 1825, 2071, Intron12 ds + 47, 2307, 2338, 2372, Intron14 as - 24, 2618, 2690, 2710, Intron19 ds + 47 and UTR3 'ds + 124 is mutated in whole or in part, with the proviso that an exchange at positions 135, 2071, 2307, 2372, 2690 and 2710 is always in Combination with at least one of the other mentioned is present. In particular, it is a sequence that completely or partially the mutations G → A (Ala, position 135), G → A (position Intron2 ds + 9), C → T (Phe, position 447), T → C (Val to Ala, position 785), G → A (Asp to Asn, position 799), G → A (Thr, position 885), G → C (Trp to Cys, position 972), C → T (Thr to Ile, position 1013), T → A (Cys to Ser, position 1825), G → A (Gly to Ser, position 2071), C → T (position Intron12 ds + 47), T → G (Leu, position 2307), A → T (Lys to stop, position 2338), A → T (Tyr to Phe, position 2372), G → A (position Intron14 as - 24), G → A (Arg to Gln, position 2618), G → A (Arg for Gln, position 2690), C → G (Ser, position 2710), C → T (position Intron19 ds + 47) and A → G (position UTR3 'ds + 124) contains ( Fig. 1, 2a to 2k), the above limitation applies.

The following sequences (haplotypes) are particularly important:

• - sequence with the mutations 135 A and 2307 T,
• - sequence with the mutations 135 A and 2307 G,
• - sequence with the mutations 135 G and 2307 G,
• - Sequence with the mutations 135 A, 2071 G, 2307 T and 2710 C.
• - Sequence with the mutations 135 A, 2071 G, 2307 G and 2710 C.
• - Sequence with the mutations 135 G, 2071 G, 2307 G and 2710 C.

The invention further relates to a method for determining Disease disposition, with all sequences and variants of human RET Protooncogene based on base exchanges in at least one of the following positions 135, Intron2 ds + 9, 447, 785, 799, 885, 972, 1013, 1825, 2071, Intron12 ds + 47, 2307, 2338, 2372, Intron14 as - 24, 2618, 2690, 2710, Intron19 ds + 47 and UTR3 'ds + 124 genotyped is compared and subsequently compared with the reference DNA sequence of the RET protooncogene , from the single mutation to all possible combinations of all variants (including any absolute number of variants that are included can) be genotyped and the corresponding statements about Allow disease disposition.

The method is characterized in that the DNA of a subject is isolated and genotyped at least at one of the exchanged positions and subsequently with the Reference DNA sequence is compared. Embodiments are preferred in which at least position 135, at least the two positions 135 and 2307 or the four Positions 135, 2071, 2307 and 2710 or each additional position separately with the four the latter positions are genotyped.  

Genotyping is carried out by sequencing or by other methods for the Detection of point mutations are suitable. This includes PCR-based Genotyping procedures such as B. allele-specific PCR, other genotyping methods using oligonucleotides (examples would be 'dot blotting', or 'oligonucleotides Ligation Assays '(OLA)), method using restriction enzymes, and' Single Nucleotide Polymorphism '(SNP) analysis using' Matrix-assisted Laser Desorption / Ionization Mass Spectrometry (MALDI), as well as in principle any available in the future Method for variant detection including chip technology in all of its technological designs.

Based on this, the method according to the invention for determining a broad Suitable for a wide range of diseases.

In one embodiment, there is a genetic predisposition to Hirschsprung's disease (congenital absence of nerve cells in the intestinal wall). There is according to the invention depending on the homozygous or heterozygous detection certain haplotypes or their combination the possibility of a differentiated statement to genetic predisposition for a short or long distance aganglionic segment to meet. Through this procedure, a genetic advice based on molecular genetics becomes available enables.

In an extension of the first variant, the procedure allows the genetic Characterization of a subpopulation within a patient group that is clinically characterized by the Occurrence of constipation is to be described, but because of patho that has not yet been objectifiable morphological or functional deficits are classified as 'idiopathic'. For the ent according to the procedure, genetically characterized subpopulation is the indication to discuss surgical therapy based solely on the genetic finding.

Furthermore, the detection of a specific sequence variant of the RET protooncogene with the Occurrence of medullary thyroid carcinoma with high penetrance in the context of a multiple endocrine neoplasia syndrome type 2A or a familial medullary Thyroid cancer associated. The detection of these mutations thus represents the is the diagnostic basis for preventive surgical intervention.

Another important object of the invention is the use of the claimed Sequence variants for the prediction of central respiratory regulation disorders with subclinical Appearance. After detection of certain mutations in the RET proto-oncogene the disturbances of the central respiratory drive in the sleep laboratory can be objectified and Define appropriate preventive measures under stressful situations.  

In the same sense, a genetic predisposition to the occurrence of an Undine syndrome (complete failure of central breathing regulation). These In this respect, the variant of embodiment acquires a special meaning, as well as known ones epidemiological risk factors for the first time genetic risk factors for the sudden Child death can be definable.

Based on the assumption that certain sequence variants of the RET protooncogene with are associated with a reduced gene activity and accordingly different Cause clinical pictures (Hirschsprung's disease, idiopathic constipation), these sequence variants represent the basis for the selection of a population for which one pharmacological regulation of gene activity may be conceivable.

The scope of the claimed invention is detailed below. To Development of the invention was the known coding DNA sequence of the human RET protooncogene including its exon flanking intron sequences in patients and Controls were investigated using direct DNA sequencing, and initially a number of genetic variants identified. In the coding region became the existing one At the time, nine new variants were discovered, seven of which have a strict association have certain clinical pictures. In addition, five were considered polymorphisms characterized variants identified in the exon-flanking regions of the gene.

Summary of the newly identified variants

Obtain the specified nucleotide positions with the associated base substitutions refer to the published sequences of the RET protooncogene with its exon-flanking Regions (Takahashi M. et al., Oncogene 3 (5): 571-578 (1988) [Acc.-No. M57464]; Checcherini I. et al., Oncogene 9 (10): 3025-3029 (1994); Myers S.M. et al., Oncogene 10 (11): 2039-2045 (1995).

Mutations

447 C → T 785 T → C 799 G → A 885 G → A 972 G → C 1013 C → T 1825 T → A 2338 A → T 2618 G → A

Polymorphisms

Intron2 ds + 9 G → A Iniron12 ds + 47 C → T Intron14 as - 24 G → A Intron19 ds + 47 C → T UTR3 'ds + 124 A → G

These variants are clearly shown in Fig. 1, 2a to 2k.

Correlations of polymorphisms with clinically relevant clinical pictures

In addition to the above newly identified sequence variants of the human RET protooncogene there are already a number of known variants, of which in connection with the invention four polymorphisms (135 G → A, Ala; 2071 G → A, Gly to Ser; 2307 T → G, Leu and 2710 C → G, Ser) were considered. Sequence variant 135 A has been shown to be strict Has association with Hirschsprung's disease. This applies in particular to the situation when the exchange is detected on both alleles. While in a healthy Control population the variant 135 A only in 5.8% (9 of 156) of the individuals on both Alleles were detectable, these were found in the patient population in 54.8% (34 of 62). A The starting point of the invention is that further sequence variants (2071 G, Gly; 2307 G and 2710 C) have a significant association with the phenotype of Hirschsprung's disease. By combining these sequence variants, haplotypes can now be defined, one diagnostic value with regard to a genetic predisposition for the disease Hirschsprung and possible associated clinical pictures. Analysis of the phenotypes shows that the defined haplotypes almost without exception with the form of short distances aganglionic segments correlated. With the exception of polymorphism 2071 G → A, Gly to Ser the sequence variants do not lead to an amino acid exchange  assumed that the variants have a direct or indirect influence on the expression exercise the gene.

Detection of specific RET haplotypes consisting of two variants

(Positions 135 G → A, Ala and 2307 T → G, Leu)
Combination 1: 135 A and 2307 T.
Combination 2: 135 A and 2307 G
Combination 3: 135 G and 2307 G

The invention is based on the concept that for the manifestation of Hirschsprung's disease the detection of a certain haplotype on an allele is not the only important factor. Disease-causing gene inactivation only becomes relevant in terms of etiology when Characteristic haplotypes occur genotypically combined. Accordingly, the homozygous combination 2 the greatest predictive value with regard to the expression of a Hirschsprung's disease, which is characterized by a short aganglionic segment. she will therefore as a preferred combination for the development of therapeutic agents, for the development of diagnostic kits, etc. used.

Detection of specific RET haplotypes consisting of four variants

(135 G → A, Ala; 2071 G → A, Gly to Ser; 2307 T → G, Leu and 2710 C → G, Ser)
Combination 1: 135 A, 2071 G, 2307 T and 2710 C.
Combination 2: 135 A, 2071 G, 2307 G and 2710 C.
Combination 3: 135 G, 2071 G, 2307 G and 2710 C.

Taking into account the principles listed in the detection of two variants, at the detection of four variants an increase in the information regarding a genetic Predisposition to Hirschsprung's disease and possible associated clinical pictures be achieved.

Correlations of mutations with clinically relevant clinical pictures

Furthermore, one known and two new mutations were identified in the coding region of the human RET protooncogene, which are located in the intracellularly located tyrosine kinase domain (2338 A → T, Lys to Stop; 2618 G → A, Arg to Gln; 2690 G → A, Arg to Gln) and a strict association with Hirschsprung's disease. These mutations are of particular importance because they show an association with a long aganglionic segment and a disturbance of the central respiratory regulation. These haplotype mutations are believed to be linked to the variant at position 135G. Based on this, it follows that, according to the observations on the second allele, the non-mutated variant of positions 2338, 2618 and 2690 is coupled to variant 135 A in the haplotype. The following combinations of haplotypes result:
Combination 1: 1. Alleles: 135 G and 2338 T (Stop) / 2. Alleles: 135 A and 2338 A (Lys)
Combination 2: 1st allele: 135 G and 2618 A (Gln) / 2. Alleles: 135 A and 2618 G (Arg)
Combination 3: 1. Alleles: 135 G and 2690 A (Gln) / 2. Alleles: 135 A and 2690 G (Arg)

The invention relates to the characterization of sequence variants of human RET Protooncogen, which in certain allele combinations according to the above Exemplary embodiments with a phenotypic expression of Hirschsprung's disease are associated with a long aganglionic segment and possible disorders of the central breathing regulation is characterized. The allele combinations mentioned have only exemplary meaning and are analogous to other mutations in the intracellular Tyrosine kinase domain transferable.

Furthermore, four new ones were created in the coding region of the human RET protooncogene Mutations identified that are located in the extracellularly located receptor domain (785 T → C, Val to Ala; 799 G → A, Asp to Asn; 972 G → C, Trp to Cys; 1013 C → T, Thr to Ile) and a strict association with Hirschsprung's disease, whereby the aganglionär Segment of different lengths can be pronounced and no disturbance of the central Breath regulation is detectable. For those located in the extracellular receptor domain Hirschsprung's disease will mutate regardless of the variant at position 135 G → A pronounced. In contrast, there is a gender dependency with an association of Disease expression of the male gender.

Furthermore, a new mutation was identified in the coding region of the human RET protooncogene, which is located in the extracellular cysteine-rich region (1825 T → A, Cys to Ser) and shows an association with Hirschsprung's disease. This mutation is of particular importance because it causes a previously unknown amino acid substitution of one of the well-known highly conserved cysteine residues and this exchange is associated with the highly penetrant form of multiple endocrine neoplasia syndrome type 2A or a familial medullary thyroid carcinoma. Both the mutated and the non-mutated variant at position 1825 are coupled in the haplotype with the variant at position 135 A. This results in the following combination of haplotypes:
Combination 1: 1. Alleles: 135 A and 1825 A (Ser) / 2. Alleles: 135 A and 1825 T (Cys)

The invention relates to the characterization of sequence variants of the human RET protooncogene, which in certain allele combinations according to the above Embodiment with a phenotypic expression of Hirschsprung's disease and a genetic predisposition with high penetrance for multiple endocrine neoplasia Type 2A syndrome or familial medullary thyroid carcinoma is associated. Here the allele combination has only exemplary meaning and is analogous to others Mutations in the extracellular cysteine-rich region are transferable.

In conclusion, the present invention turns into a new genetic inheritance model proposed for Hirschsprung's disease. While so far a dominant negative Mechanism of action of those demonstrated in patients with Hirschsprung's disease Mutations (approximately 20% of patients) has been postulated, it is shown that it is in human RET proto-oncogene other genetic traits such as the polymorphism at position 135 G → A gives that through another mechanism of action (e.g. autosomal recessive) Condition of the disease. According to the observations made within the Invention is characterized by the polymorphism position 135 both the phenotypic Severity of the disease in patients with a proven mutation in RET Protooncogene modified as well as by the homozygous occurrence of the variant position 135 A per se the disease is pronounced with a weak phenotype. This inheritance model can be followed by the invention for 65 to 70% of patients with a disease Hirschsprung's genetic traits are defined in the RET proto-oncogene, which are responsible for the Are responsible for the severity of the disease.

The invention is explained in more detail by an embodiment.

material and methods

The direct DNA sequencing method according to Sanger was used to collect the entire polymorphic spectrum of the human RET protooncogene. For this purpose, all 21 exons known to date with the associated exon-flanking intron sequences were amplified by means of PCR. These PCR fragments were purified on the agarose gel, cut out and eluted with sterile water. Sequencing was carried out using the Thermo Sequenase ^™ Fluorescent Cycle Sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany). For the Sequenzieung the PCR primers were used identical Cy5 ^™ -labeled primer sequence analysis so that by means of ALF express (Pharmacia Biotech, Freiburg, Germany) using polyacrylamide gels (1 M urea) was performed. In the example, the detection of specific RET haplotypes consisting of four variants is carried out.

The specific conditions for the amplification were as follows:
For exon 2 (position 135), the forward primer RETE2S1 with the sequence 5'-CCATATTCTCACCATCCCTC-3 'and the reverse primer RETE2A1 with the sequence 5'-AGTGTCAGCGGCTGTGATAA-3' (Cy5 ^™ -labeled) were used. Exon 11 was amplified using the two primers RETE11S5: 5'-CCTCTGCGGTGCCAAGCCTC-3 'and RETE11A5: 5'-CCTCGTCTGCCCAGCGTTG-3' (Cy5 ^™ -labeled). For exon 13, the two primers RETE13S: 5'-GCAGGCCTCTCTGTCTGAACTT-3 'and RETE13A: 5'-GGAGAACAGGGCTGTATGGA-3' (Cy5 ^™ -labeled). For exon 15, the two primers RETE15S2: 5'-CCCCCGGCCCAGGTCTCAC-3 'and RETE15A2: 5'-GCTCCACTAATCTTCGGTATCTTT-3' (Cy5 ^TM -labeled). The reaction conditions for these four fragments were as follows: 50 ng genomic DNA, dNTP 200 mM, 200 nM each primer, 10 × PCR buffer and 1 U AmpliTaq (both from PE Applied Biosystems, Weiterstadt, Germany). The magnesium chloride concentration varied as follows: exon 13 2 mM, exon 2, exon 11 and exon 15 each 3 mM. The reaction batches were 25 ul each. The amplification was carried out with the following temperature profile:
Exon 2: 94 ° C for 4 min; 35 cycles: 94 ° C 30 sec, 57 ° C 30 sec, 72 ° C 30 sec and finally 72 ° C 10 min
Exon 11: 94 ° C for 4 min; 35 cycles: 94 ° C 30 sec, 65 ° C 30 sec, 72 ° C 30 sec and finally 72 ° C 10 min
Exon 13: 94 C 4 min; 35 cycles: 94 ° C 30 sec, 60 ° C 30 sec, 72 ° C 30 sec and finally 72 ° C 10 min
Exon 15: 94 C 4 min; 35 cycles: 94 ° C 30 sec, 55 ° C 30 sec, 72 ° C 30 sec and finally 72 ° C 10 min

The sequencing was carried out using the Thermo Sequenase ^TM cycle sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany). The Cy5 ^™ -labeled PCR primers described above were used as sequencing primers.

All PCR and sequencing reactions were carried out in a GeneAmp® PCR System 9700 (PE Applied Biosystems, Weiterstadt, Germany).

The products of the sequence reactions were on a 300 micron thick polyacrylamide gel (1 M urea) on the A. L. F. express (Pharmacia Biotech, Freiburg, Germany) separated and the sequence variants compared to the normal sequence detected.

The sequence variants considered in the exemplary embodiment in any case change the interface of a restriction endonuclease, so that the detection of the variants was also carried out by means of RFLP analysis. The fragments were amplified using the above-mentioned primer pairs. The PCR products were also purified. 5 µl of PCR product in a total volume of 10 µl was used for digestion. The following restriction endonucleases were used: exon 2 (position 135) EagI, exon 11 (position 2071) BanI, exon 13 (position 2307) TaqI and exon 15 (position 2710) RsaI (all enzymes from New England Biolabs, Schwalbach, Germany). The incubation period was five hours. The following fragments were detected:

The fragments were separated on a 500 μm thick polyacrylamide gel (1 M Urea) by means of A.L.F.express (Pharmacia Biotech, Freiburg, Germany).

literature

Takahashi M., Buma Y., Iwamoto T., Inaguma Y., Ikeda H., Hiai H .: Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 3 (5): 571-578 (1988)
Checcherini L, Hofstra RMW, Luo Y., Stulp RP, Barone V., Stelwagen T., Bocciardi R., Nijveen H., Bolino A., Seri M., Ronchetto P., Pasini B., Bozzano M., Buys CH. HCM, Romero G .: Dna polymorphism and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene. Oncogene 9 (10): 3025-3029 (1994)
Myers SM, Eng Ch., Ponder BAJ, Mulligan LM: Characterization of RET proto oncogene 3 'splicing variants and polyadenylation sites: a novel C-terminus for RET. Oncogene 10 (11): 2039-2045 (1995).

Claims (29)

1. Sequence of the human RET protooncogene, characterized in that the bases at positions 135, Intron2 ds + 9, 447, 785, 799, 885, 972, 1013, 1825, 2071, Intron12 ds + 47, 2307, 2338, 2372 , Intron14 as - 24, 2618, 2690, 2710, Intron19 ds + 47 and UTR3 'ds + 124 are exchanged in whole or in part, with the proviso that an exchange at positions 135, 2071, 2307, 2372, 2690 and 2710 always in combination with at least one of the other mentioned. 2. Sequence according to claim 1, characterized in that it is wholly or partly the Base exchanges G → A (position 135), G → A (position Intron2 ds + 9), C → T (position 447), T → C (position 785), G → A (position 799), G → A (position 885), G → C (position 972), C → T (Position 1013), T → A (position 1825), G → A (position 2071), C → T (position Intron12 ds + 47), T → G (position 2307), A → T (position 2338), A → T (position 2372), G → A (position Intron14 as - 24), G → A (position 2618), G → A (position 2690), C → G (position 2710), C → T (position Intron19 ds + 47), and A → G (position UTR3 'ds + 124), with the proviso that a Exchange at positions 135, 2071, 2307, 2372, 2690 and 2710 always in combination with at least one of the other mentioned is present. 3. Sequence according to claim 1 and 2, characterized by the mutations 135 A and 2307 T. 4. Sequence according to claim 1 and 2, characterized by the mutations 135 A and 2307 G. 5. Sequence according to claim 1 and 2, characterized by the mutations 135 G and 2307 G. 6. Sequence according to claim 1 and 2, characterized by the mutations 135 A, 20710, 2307 T and 2710 C. 7. Sequence according to claim 1 and 2, characterized by the mutations 135 A, 2071 G, 2307 G and 2710 C. 8. Sequence according to claim 1 and 2, characterized by the mutations 135 G, 2071 G, 2307 G and 2710 C. 9. A method for determining disease dispositions, characterized in that the DNA of a subject isolated and the sequence of the human IET protooncogene based base exchange at at least one of the following positions 135, Intron2 ds + 9, 447, 785, 799, 885, 972, 1013, 1825, 2071, Intron12 ds + 47, 2307, 2338, 2372, Intron14 as - 24, 2618,  2690, 2710, Intron19 ds + 47 and UTR3 'ds + 124 is genotyped and subsequently with the Reference DNA sequence of the RET protooncogene is compared 10. The method according to claim 9, characterized in that all possible combinations from variants from the single mutation to all possible combinations of all variants including any absolute number of variants. 11. The method according to claim 9 or 10, characterized in that the DNA of a subject isolated and genotyped at least at position 135. 12. The method according to claim 9 or 10, characterized in that the DNA of a subject isolated and genotyped at least at the 2 positions 135 and 2307. 13. The method according to claims 9 or 10, characterized in that the DNA of a subject isolated and genotyped at least at the 4 positions 135, 2071, 2307 and 2710. 14. The method according to claim 9, 10 or 13, characterized in that the positions 135, 2071, 2307 and 2710 can be genotyped. 15. The method according to claim 14, characterized in that at least 3 of the 4 positions 135, 2071, 2307 and 2710 can be genotyped. 16. A method for determining disease dispositions 9, 10 or 15, thereby characterized in that positions 135, 2307 and 2710 are genotyped. 17. The method according to any one of claims 9 to 16, characterized in that the Genotyping by sequencing or by other methods used for the detection of Variants are suitable. 18. The method according to any one of claims 9 to 17 for determining a disposition any form of defecation disorders, in particular the Hirschsprung motor bus or the idiopathic constipation, to determine a disposition for multiple endocrine Type 2A neoplasia syndrome or familial medullary thyroid carcinoma Determination of a disposition for central respiratory regulation disorders, especially the undine- Syndrome or sudden infant death. 19. The method according to any one of claims 9 to 17 for determining an individual different functional state of the intestinal neural system, especially in Association with any form of constipation.   20. The method according to any one of claims 9 to 17 for determining the genetic Predisposition and the severity of the phenotypic manifestation of diseases, preferably as listed in claim 19, preferably Hirschsprung's disease. 21. The method according to claim 11, characterized in that the detection of homozygous Variant of position 135 A as the sole characteristic or in association with any one other genetic trait is a sufficient criterion for the phenotypic association is used with a Hirschsprung's disease characterized by a short aganglionic segment is characterized. 22. The method according to claim 9 or 10, characterized in that the detection of a heterozygous mutation in the intracellular region of the coding sequence of the RET Protooncogen in particular position 2338, 2618 and 2690, which is combined with the variant position 135 A on the respective other allele, as a sufficient criterion for the phenotypic Association with Hirschsprung's disease and possibly disorders of the central one Respiratory regulation is used, which is characterized by a long aganglionic segment and 23. The method according to claim 9 or 10, characterized in that the detection of a heterozygous mutation in the extracellular region of the coding sequence of the RET Protooncogen in particular positions 785, 799, 972 and 1013 regardless of the variant of both Alleles at position 135, but found gender penetrance as sufficient criterion for the phenotypic association with Hirschsprung's disease is used, which is characterized by an aganglionic segment of different lengths. 24. The method according to claim 9 or 10, characterized in that the detection of a heterozygous mutation in the cysteine-rich region of the RET protooncogene in particular position 1825, which is combined with the position 135 A variant on both alleles, as sufficient Criterion for phenotypic association with Hirschsprung's disease and one genetic predisposition for the manifestation of multiple endocrine neoplasia syndrome Type 2A or familial medullary thyroid cancer is used. 25. Use of the exchanged sequence variants mentioned in claim 9 Development of a new class of therapeutics based on the human RET proto-oncogene are aimed at the 5 'regulatory area or in the promoter area and via Regulation of transcription and translation and by influencing their efficiency, primarily by regulating expression.   26. Use according to any one of claims 9 to 17 to optimize the individual on the ret receptor tyrosine kinase and its gene-directed therapy or intervention. 27. Use of the exchanged sequence variants mentioned in claim 9 for the construction of genes and vectors. 28. Use according to claim 25 to 27 for the development of a diagnostic kit, or any method of genotyping. 29. Use according to claim 1 to 8 and 9 to 28 for the development of in vitro and in vivo test systems that express individual forms of the human RET protooncogene, the test systems for examining the pathophysiology of diseases of general medically important characteristics with participation of the RET protooncogene serve, as well as for Development and testing of individually specific therapeutic agents in general.