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WO2005078132A2 - OLIGONUCLEOTIDE PROBES FOR DIAGNOSIS AND IDENTIFICATION OF THE DIFFERENT FORMS OF β-THALASSEMIA, METHODS AND DIAGNOSTIC KIT THEREOF - Google Patents

OLIGONUCLEOTIDE PROBES FOR DIAGNOSIS AND IDENTIFICATION OF THE DIFFERENT FORMS OF β-THALASSEMIA, METHODS AND DIAGNOSTIC KIT THEREOF Download PDF

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WO2005078132A2
WO2005078132A2 PCT/IT2005/000066 IT2005000066W WO2005078132A2 WO 2005078132 A2 WO2005078132 A2 WO 2005078132A2 IT 2005000066 W IT2005000066 W IT 2005000066W WO 2005078132 A2 WO2005078132 A2 WO 2005078132A2
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seq
ivs
agg
cct
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WO2005078132A3 (en
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Aurelio Maggio
Antonino Giambona
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BIOCHEMICAL SYSTEMS INTERNATONAL Srl
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to oligonucleotide probes for diagnosis of the different forms of ⁇ -thalassemia and methods and diagnostic kit thereof. More in particular, the invention refers to the oligonucleotide sequences to be used as probes and/or as specific primers for the diagnosis and discrimination of the different forms of ⁇ -thalassemia and methods and diagnostic kit thereof.
  • Thalassemia is a genetic disease which belongs to the hemoglobinopathies, namely the hereditary diseases involving hemoglobin (Hb), the main erythrocytary component.
  • hereditary microcytic hypochromic anemias in the most cases due to a quantitative defect in the synthesis of one or more of the hemoglobin polypeptidic chains ⁇ ( ⁇ - thalassemia) or ⁇ ( ⁇ -thalassemia).
  • ⁇ or ⁇ chains were synthesized in a coordinate way and the unbalanced production of a globin chain causes a serious erytrocyte damage which results in a block of the production of normal ⁇ 2 or ⁇ 2 hemoglobin tetramers or in an intracellular accumulation of free chains into erythroblasts.
  • ⁇ -thalassemia is the more frequent and more important genetic disease from the sanitary point of view in the Mediterranean area (Sicily, Campania, Sardinia, Calabria, Puglia, North Italy regions for emigrants from South to North).
  • ⁇ -thalassemias can be classified in two main groups: ⁇ ° and ⁇ + thalassemia.
  • ⁇ °-thalassemias are characterized by the presence of the mutated gene which does not produce globin, while ⁇ + -thalassemias present a partial synthesis of ⁇ -globin.
  • ⁇ + -thalassemias can be in their turn classified in serious ⁇ + if the synthesis is very reduced, in minor ⁇ + if the globin synthesis is more marked and in silent ⁇ + if the synthesis is very evident.
  • the classification of the different forms of thalassemia is connected with the type of mutation involving the ⁇ -globin gene.
  • ⁇ °-thalassemias present the respective molecular defects: - point mutation into the exon which encodes a STOP codon, which results in the synthesis of a truncated globin chain.
  • the most common mutation present in Sicily and in the Mediterranean area is at Cd39 level.
  • mutations at the level of introns such as I VS1 , nt5, IVS1 , nt6 which determine a minor ⁇ + phenotype since the mutation estabilishes an alternative splicing site which is used less frequently in comparison to the normal site. Beside the minor ⁇ + -thalassemias ther are also the silent ⁇ + - thalassemias wherein the phenotype in a subject under study presents one or more normal hematological and/or hemoglobin parameters. This is the case of the mutation -101 at the promoter level or of the mutation
  • IVS2, nt 844 The pathogenetic mechanisms of ⁇ + -thalassemic syndromes differ from those typical of many forms of ⁇ -thalassemia, often associated with a gene deletion in the locus of the alfa globin chain structural gene. In fact, as previously described, in the most common forms of ⁇ + -thalassemia the globin gene is normally transcribed but the elaboration is defective.
  • the point mutations can also map at the level of ⁇ globin locus or involve the amount of y chain to be synthesized (the three genes ⁇ , ⁇ , y are localized on the same chromosome 11 )(1 ).
  • the first group which comprises mutations ⁇ 39 , IVS 1 ,110 and IVS 1 ,6 recurring with a frequency of 71 %
  • the second group which comprises mutations -87, ⁇ s , IVS 1 ,1 and IVS 2, 745 recurring with a frequency of 20%.
  • the gene typization of genomic DNA samples can be carried out by various methods in molecular biology, some of them involving the hybridization with DNA oligonucleotidic probes complementary to the mutated sequences to be analysed.
  • RDB Reverse Dot Blot
  • the RDB method is based on the hybridization reaction between an aminomodified oligonucleotide, anchored at the 5' end to a nylon membrane activated by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide through amidic bond and the target genomic DNA amplified by PCR in the presence of a biotinilated nucleotide (dUTP- biotin).
  • dUTP- biotin biotinilated nucleotide
  • Streptavidin molecules link biotin incorporated into amplified DNA, while the alkaline phosphatase reacts with a chromogen mixture of NBT and BICP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indoylphosphate, respectively).
  • chromogens act as substrates for the colorimetric reaction which leads to the formation of an insoluble blu precipitate (2,3).
  • the oligonucleotides complementary to the normal sequences, as well as the oligonucleotides complementary to the mutated sequences were placed on the strips, in such a way to test whether the patient is heterozygote or homozygote for the tested mutation.
  • the RDB hybridization shows some positive characteristics in comparison with other techniques involving the hybridization with DNA probes.
  • the first main advantage is undoubtely the use of the sequences unlabelled with radioisotopes which makes this kind of analysis sure and easy to carry out also in not equipped radioisotope laboratories.
  • the RDB allows testing a certain number of samples simultaneously for many gene mutations (from 1 to 50 and over) and this results in a remarkable saving of time (about 6 hours).
  • the "Dot Blot" technique used in the past, employed a radioactive for the signal detection, thus allowing a limited number of samples to be tested for one mutation over a long period of time (about 24 hours).
  • the oligonucleotides complementary to the mutated gene sequences and to the respective normal sequences to be employed as probes in the RDB technique there is the need to meet some requirements, for example, the specific hybridization of the oligonucleotides fixed on the same blot strip with the target sequences (normal and mutated) has to be carried out at the same temperature. It is also required the avoidance of probes circularization or the heterodimer formation that would make the probes hybridization and subsequent use ineffective.
  • sequences according to the invention can be advantageously used for the preparation of diagnostic kits for ⁇ -thalassemia without the limits of the known methodologies.
  • oligonucleotide probes able to hybridize specifically with both mutated and normal target sequences that are present at genomic DNA level to be tested, were designed by the inventors. Said probes show several advantages in terms of specificity and effectiveness of the hybridization process, as well as the chance to recycle the same probe. They are not able to work in the same conditions required for hybridization and washing, thus avoiding to obtain false positives and false negatives.
  • the advantageous characteristics of the oligonucleotide probes according to the present invention are the following: the oligonucleotide lenght being comprised between 14 and 22 bases in such a way that the denaturation temperature is between 46°C and 50°C; the central localization of the mutated nucleotide; a balanced ratio between the number of guanine and cytosine bases and the number of thymine and adenine bases; the lack of circularization phenomena and/or loops or heterodimers formation which allows the possible effective use in a subsequent hybridization reaction .
  • the probes and primers for the identification of the mutations associated with ⁇ -thalassemia in the Mediterranean area indicated as follows, were designed:
  • Figure 2 shows the localization of the above mentioned mutations at level of ⁇ globin gene. Therefore, it is an object of the present invention oligonucleotide probes able to hybridize to the target sequence belonging to the ⁇ globin gene that may comprise the sequences:
  • CA ATC CAG CTA CCA TTC (SEQ ID No 11 );
  • AC CCT AGG TTG TGG CT (SEQ ID No 25); AC CCT AGG ATG TGG CT (SEQ ID No 26) ;
  • CA ACC CTA TGG TGT GG (SEQ ID No 30); CT GGG CAT AAA AGT CAG (SEQ ID No 31 );
  • the target sequence belonging to the ⁇ -globin gene may comprise at least one mutated sequence selected from the group consisting of:
  • oligonucleotide probes according to the present invention can be chemically modified and/or labelled, said probes being modified at the 5' end with an aminic group or with a poli-T sequence.
  • the aminomodified or differently derivatized oligonucleotide probes can be anchored to a solid support, preferably to an opportunely activated membrane, negatively or positively charged.
  • the membrane is negatively charged and is preferably activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
  • a positively charged membrane as a solid support would be necessary.
  • an opportunely activated solid support comprising at least one of the above oligonucleotide probes anchored thereon.
  • said solid support can be an opportunely activated membrane with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
  • the oligonucleotide probes can be alternatively labelled with at least one fluorochrome or a radioactive isotope at the 3' and/or 5' ends of the oligonucleotide sequence.
  • the fluorochrome may be an emission and/or excitation fluorochrome, preferably it is fluorescein, for the detection, for example, by PCR Real time.
  • oligonucleotide sequences according to the present invention for the diagnosis and the identification of the ⁇ -globin gene mutations associated with ⁇ -thalassemia, in heterozygote and homozygote subjects.
  • the present invention concerns a method for the detection and the identification of the ⁇ -globin gene mutations associated with ⁇ -thalassemia, in heterozygote and homozygote subjects, comprising the following steps: a) DNA extraction from the biological sample (any organic tissue: blood, amniotic liquid, chorial villi liquid) under examination; b) PCR amplification of ⁇ -globin gene containing the target sequence through the use of a set of flanking primer of the ⁇ -globin gene; c) detection of the mutated target sequence and/or the corresponding unmutated sequence amplified in step b) through the use of oligonucleotide probes according to the present invention able to hybridize themselves to said target sequence belonging to the ⁇ -globin gene.
  • the set of primer of step b) of the above method may comprise at least one pair of primer selected from the group of oligonucleotide sequences:
  • AGA GAT ATA TCT TAG AGG GAG (SEQ ID No 50); GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51 ); CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52);
  • the mutated target sequence of step c) of the above method may comprise a sequence with at least one mutation, said mutation being selected from the group consisting of:
  • the oligonucleotide probes used in step c) of the method according to the present invention can be anchored to a solid support.
  • Said solid support may be a negatively or positively charged membrane, opportunely activated. Said activation can be carried out with 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide in the case of a negatively charged membrane to be employed as a solid support.
  • a diagnostic kit for the detection and the identification of the ⁇ -globin gene mutations associated with ⁇ -thalassemia, in heterozygote and homozygote subjects which may comprise: a) a set of primer for the PCR amplification of ⁇ -globin gene containing the target sequence, which may comprise at least two among the following oligonucleotide sequences: AGA GAT ATATCT TAG AGG GAG (SEQ ID No 50);
  • TCATTC GTC TGT TTC CCATTC TAAAC SEQ ID No 53
  • TGC ATATTC ATAATC TCC CTA CTT T SEQ ID No 54
  • the mutations detected and identified by the diagnostic kit according to the present invention may be selected from the group consisting in:
  • the oligonucleotide probes of the diagnostic kit according to the present invention can be anchored to a solid support.
  • Said solid support can be a positively or negatively charged membrane, opportunely activated. Said activation can be carried out with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the case of a negatively charged membrane to be employed as a solid support.
  • Further object of the present invention are primers for the PCR amplification of the ⁇ -globin gene that may comprise at least one of the sequences: AGA GAT ATA TCT TAG AGG GAG (SEQ ID No 50);
  • object of the present invention are antisense oligonucleotides that may comprise at least one of the sequences:
  • CA ATC CAG CTA CCA TTC (SEQ ID No 11 );
  • CT GGG CAT AAA AGT CAG (SEQ ID No 31 );
  • RDB Reverse Dot Blot assay, wherein, after the binding of the conjugated streptavidin with alkaline phosphatase (AP) to the biotin, a colorimetric reaction and the subsequent formation of an insoluble precipitate in presence of two chromogens take place;
  • Figure 2 shows the localization of the gene mutations associated with ⁇ -thalassemia, indicated with an abbreviation, at level of the ⁇ -globin gene locus; EX: exon; IVS: intron; Cd: codon; nt: nucleotide;
  • Figure 3 in panels A, B, C, D, E shows the phenotypes of the different forms of ⁇ °-thalassemia and ⁇ + -thalassemia.
  • panel A shows the phenotype of those subjects who are not carrier of ⁇ - thalassemia
  • panel B shows the phenotype of those subjects with ⁇ °- thalassemia
  • panel C shows the phenotype of those subjects with severe ⁇ + -thalassemia
  • panel D shows the phenotype of those subjects with minor ⁇ + -thalassemia
  • panel E shows the phenotype of those subjects with silent ⁇ + -thalassemia.
  • EXAMPLE 1 Reverse Dot Blot experiment using the oligonucleotide probes for the detection and the identification of gene mutations associated with ⁇ -thalassemia MATERIALS AND METHODS Reverse Dot Blot (RDB) was carried out on 10.000 DNA samples from ⁇ -thalassemia healthy carrier and from normal subjects. The results achieved during the study carried out by the authors of the present invention were showed in Table 1.
  • Table 1 shows the incidence frequency and the genetic mutation type associated with ⁇ - thalassemia, analysed in a group of patients belonging to the Sicilian population (10.000 chromosomes). Table 1
  • Amplified DNA labelling can be carried out through two way: 1) using the biotin-16-dUTP base during the course of PCR, which links randomly in place of thymine; 2) using biotinilated modified primer at 5' (primer-5'-biotin-sequence).
  • the PCR amplification conditions DNA samples to be undergone RDB analysis were reported in Table 2. Table 2
  • the oligonucleotide sequences of the primer according to the present invention that can be used for the amplification of the ⁇ -globin gene are the following: 1) AGAGAT ATA TCT TAG AGG GAG (SEQ ID No 50) 2) GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51) 3) CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52) 4) TCA TTC GTC TGT TTC CCA TTC TAA AC (SEQ ID No 53) 5) TGC ATA TTC ATA ATC TCC CTA CTT T (SEQ ID No 54) 6) CAC TGA CCT CCC ACA TTC CC (SEQ ID No 55) Subsequently, the biotinilated PCR product (biotinilation carried out by using previously biotinilated primers or by adding biotin) was hybridized (hybridization in liquid phase) with the oligonucleotide probes anchored to the membrane.
  • DNA denaturation was carried out maintaining the eppendorf tubes at a temperature of 100°C for 10 minutes. The DNA thus denaturated was transferred into the 15 ml tube containing the membrane, being careful not to pour the content directly onto the membrane. Tubes were incubated at a temperature of 45°C for 60 minutes and maintained under shaking. Membrane washing The membranes were extracted from 15 ml tubes, and collected in a becker containing about 200 ml of hybridization solution at a temperature of 45°C and were maintained under shaking for 20 minutes.
  • the streptavidin-AP solution was removed; therefore 50 ml of the hybridization solution were added and the membranes were maintained under shaking for 10 minutes at room temperature. This last operation was repeated another time. Strips were incubated with 50 ml of Genius Buffer at room temperature and maintained under shaking for 5 minutes. Also this operation was repeated again. In case wherein the streptavidin-peroxidase conjugate was used, the incubation has to be carried out twice for 5 minutes in 100 mmol/L sodium citrate pH 5.
  • streptavidin binds the amplified DNA incorporated biotin, while alcaline phosphatase will react with a chromogen containing a phosphate mixture (NBT and BICP) catalyzing the phosphate transfer reaction from BICP to NBT thus resulting in the formation of a blu insoluble precipitate at level of the probe.
  • the chromogen is a 0,1 mg/ml solution of 3 ⁇ 3',5',5',tetramethylbenzidine, 000,3% H 2 0 2 in 100 mmol/L sodium citrate pH 5.
  • the staining solution was prepared by adding 45 ⁇ l of NBT and 35 ⁇ l of BCIP to the aliquotes of 10 ml of Genius Buffer. 10 ml of staining solution were prepared for every 6 strips, because such solution must cover totally the strips, that must not overlap each other .
  • the chromogen is a 0,1 mg/ml solution of 3', 3', 5', 5', tetramethylbenzidine, 000,3% H 2 0 2, in 100 mmol/L sodium citrate pH 5.
  • the strips were incubated in the dark at room temperature until stained spots appear, for about 15-45 minutes. Finally, were washed in dH 2 0 and dried at room temperature.
  • CONCLUSIONS The Reverse Dot Blot was designed and tested to the laboratory of pre-birth diagnosis of thalassemia which is centralized to the
  • Ematologia II U.O. is the main centre of the Sicily Region for the thalassemia treatment and pre-birth diagnosis.
  • the experimentation was carried out by using the samples of normal subjects and healthy carriers of ⁇ -thalassemia with the phenotypes as described in figure 3, panels A, B, C, D, E.
  • the panel A shows the phenotype of subjects who were not carriers of ⁇ -thalassemia.
  • the MCV value is between 80 and 90 fl, while HbA2 is less than 3,5%.
  • the panel B shows the phenotype of subjects with ⁇ °- thalassemia, mainly characterized in that the MCV value is very low and HbA2 is very high.
  • the panel C shows the phenotype of subjects with severe ⁇ + - thalassemia. It is notable that the MCV value was increased in comparison to the ⁇ °-thalassemia while HbA2 value is less than 5%.
  • the panel D shows the phenotype of subjects with minor ⁇ + - thalassemia. The MCV value was increased in comparison to the severe ⁇ + -thalassemia (around 70 fl) while HbA2 value is about 4%.
  • the panel E shows the phenotype of subjects with silent ⁇ + - thalassemia. The MCV value was normal (> 80 fl) and the HbA2 value may range between 3,3% to 4%.
  • the aforesaid Reverse Dot Blot is a particularly versatile system, since the oligoprobes work at the same hybridization and washing temperature. Therefore, it is possible to manufacture kits or membrane with the probes of the present invention to be correlated to the mutations present inside a certain population (4) or to the phenotype of the subject under examination. Therefore such a system is suitable for the molecular screening of those subject who carry ⁇ -thalassemia and for the pre-birth diagnosis and may be adjusted for the analysis of other globin genes (5).
  • BIBLIOGRAPHY (1) Giambona, P. Lo Gioco, M. Marino, I. Abate, R. Di Marzo, M. Renda, F. Di Trapani, F. Messana, S.

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Abstract

The invention relates to oligonucleotidic probes for the diagnosis and the identification of gene mutations associated with β-thalassemia, methods and kits thereof.

Description

OLIGONUCLEOTIDE PROBES FOR DIAGNOSIS AND IDENTIFICATION OF THE DIFFERENT FORMS OF β-THALASSEMIA, METHODS AND DIAGNOSTIC KIT THEREOF
The present invention relates to oligonucleotide probes for diagnosis of the different forms of β-thalassemia and methods and diagnostic kit thereof. More in particular, the invention refers to the oligonucleotide sequences to be used as probes and/or as specific primers for the diagnosis and discrimination of the different forms of β-thalassemia and methods and diagnostic kit thereof. Thalassemia is a genetic disease which belongs to the hemoglobinopathies, namely the hereditary diseases involving hemoglobin (Hb), the main erythrocytary component. They are hereditary microcytic hypochromic anemias in the most cases due to a quantitative defect in the synthesis of one or more of the hemoglobin polypeptidic chains α (α- thalassemia) or β (β-thalassemia). Tipically, the α or β chains were synthesized in a coordinate way and the unbalanced production of a globin chain causes a serious erytrocyte damage which results in a block of the production of normal α2 or β2 hemoglobin tetramers or in an intracellular accumulation of free chains into erythroblasts. β-thalassemia is the more frequent and more important genetic disease from the sanitary point of view in the Mediterranean area (Sicily, Campania, Sardinia, Calabria, Puglia, North Italy regions for emigrants from South to North). β-thalassemias can be classified in two main groups: β° and β+ thalassemia. β°-thalassemias are characterized by the presence of the mutated gene which does not produce globin, while β+-thalassemias present a partial synthesis of β-globin. β+-thalassemias can be in their turn classified in serious β+ if the synthesis is very reduced, in minor β+ if the globin synthesis is more marked and in silent β+ if the synthesis is very evident. The classification of the different forms of thalassemia is connected with the type of mutation involving the β-globin gene. β°-thalassemias present the respective molecular defects: - point mutation into the exon which encodes a STOP codon, which results in the synthesis of a truncated globin chain. The most common mutation present in Sicily and in the Mediterranean area is at Cd39 level.
- point deletion or insertion inside the exon sequence (1 ,2 bases) which determines the production of an altered protein. Among these mutations we mention the deletion of the second base from codon 6, the deletion of two bases from codons 5 and 8, the deletion of one base from codons 44 and 76 or, further, the insertion of one base between codons 8 and 9.
- mutation at the level of the dinucleotides involved in the "splicing" or in the mRNA maturation which causes insertion or deletion of bases. Among these mutations we mention the base substitution at positions IVS1 , nt1 or nt2, at positions IVS2, nt1 or nt 850. Generally, serious β+ -thalassemia involve introns. The base substitution at positions IVS1 , nt 110, nt 116, IVS2, nt 745 can estabilish an alternative splicing site with the insertion of many bases. The new resulting sequence is not totally employed, thus resulting in a poor production of globin. There are other mutations at the level of introns, such as I VS1 , nt5, IVS1 , nt6 which determine a minor β+ phenotype since the mutation estabilishes an alternative splicing site which is used less frequently in comparison to the normal site. Beside the minor β+-thalassemias ther are also the silent β+- thalassemias wherein the phenotype in a subject under study presents one or more normal hematological and/or hemoglobin parameters. This is the case of the mutation -101 at the promoter level or of the mutation
IVS2, nt 844. The pathogenetic mechanisms of β+-thalassemic syndromes differ from those typical of many forms of α-thalassemia, often associated with a gene deletion in the locus of the alfa globin chain structural gene. In fact, as previously described, in the most common forms of β+-thalassemia the globin gene is normally transcribed but the elaboration is defective.
The point mutations, as well as involving the β globin locus, can also map at the level of δ globin locus or involve the amount of y chain to be synthesized (the three genes β, δ, y are localized on the same chromosome 11 )(1 ). In particular, among the Sicilian population two main β-globin gene mutation groups, occurring with a certain frequency to determine the β+-thalassemic phenotype, were identified: the first group which comprises mutations β39, IVS 1 ,110 and IVS 1 ,6 recurring with a frequency of 71 % and the second group which comprises mutations -87, βs, IVS 1 ,1 and IVS 2, 745 recurring with a frequency of 20%. The gene typization of genomic DNA samples can be carried out by various methods in molecular biology, some of them involving the hybridization with DNA oligonucleotidic probes complementary to the mutated sequences to be analysed. One of the methods more frequently used is, for example, the Reverse Dot Blot (RDB) schematically illustrated in figure 1. The RDB method is based on the hybridization reaction between an aminomodified oligonucleotide, anchored at the 5' end to a nylon membrane activated by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide through amidic bond and the target genomic DNA amplified by PCR in the presence of a biotinilated nucleotide (dUTP- biotin). At the end of the hybridization reaction between the probes and the genomic DNA, the membrane was soaked with a solution containing alkaline phosphatase conjugated streptavidin molecules. Streptavidin molecules link biotin incorporated into amplified DNA, while the alkaline phosphatase reacts with a chromogen mixture of NBT and BICP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indoylphosphate, respectively). Such chromogens act as substrates for the colorimetric reaction which leads to the formation of an insoluble blu precipitate (2,3). Also the oligonucleotides complementary to the normal sequences, as well as the oligonucleotides complementary to the mutated sequences, were placed on the strips, in such a way to test whether the patient is heterozygote or homozygote for the tested mutation. The RDB hybridization shows some positive characteristics in comparison with other techniques involving the hybridization with DNA probes. The first main advantage is undoubtely the use of the sequences unlabelled with radioisotopes which makes this kind of analysis sure and easy to carry out also in not equipped radioisotope laboratories. Furthermore, the RDB allows testing a certain number of samples simultaneously for many gene mutations (from 1 to 50 and over) and this results in a remarkable saving of time (about 6 hours). Instead, the "Dot Blot" technique, used in the past, employed a radioactive for the signal detection, thus allowing a limited number of samples to be tested for one mutation over a long period of time (about 24 hours). For the manufacture of the oligonucleotides complementary to the mutated gene sequences and to the respective normal sequences to be employed as probes in the RDB technique, there is the need to meet some requirements, for example, the specific hybridization of the oligonucleotides fixed on the same blot strip with the target sequences (normal and mutated) has to be carried out at the same temperature. It is also required the avoidance of probes circularization or the heterodimer formation that would make the probes hybridization and subsequent use ineffective. In the light of the above, it is evident the need to dispose of new biotechnological instruments, such as oligonucleotide probes and specific primer, to be used in the recent biomolecular techniques for the diagnosis, the identification and the differentiation of some forms of β-thalassemia, also as regards homozygosis/heterozygosis, which allow to overcome the limits of the adopted techniques so far. The authors of the present invention have now found specific gene oligonucleotide sequence groups to be employed as hybridization probes and/or PCR primers, for the diagnosis, differentiation and identification of some forms of β-thalassemia, including the possible homozygosis/heterozygosis of the subject. The sequences according to the invention can be advantageously used for the preparation of diagnostic kits for β-thalassemia without the limits of the known methodologies. In the course of the present study, after identifying the mutation inside the gene map, several oligonucleotide probes, able to hybridize specifically with both mutated and normal target sequences that are present at genomic DNA level to be tested, were designed by the inventors. Said probes show several advantages in terms of specificity and effectiveness of the hybridization process, as well as the chance to recycle the same probe. They are not able to work in the same conditions required for hybridization and washing, thus avoiding to obtain false positives and false negatives. The advantageous characteristics of the oligonucleotide probes according to the present invention are the following: the oligonucleotide lenght being comprised between 14 and 22 bases in such a way that the denaturation temperature is between 46°C and 50°C; the central localization of the mutated nucleotide; a balanced ratio between the number of guanine and cytosine bases and the number of thymine and adenine bases; the lack of circularization phenomena and/or loops or heterodimers formation which allows the possible effective use in a subsequent hybridization reaction . In particular, the probes and primers for the identification of the mutations associated with β-thalassemia in the Mediterranean area, indicated as follows, were designed:
Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6 T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92
OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8 (-AA) ; Cd 8/9 (+G) ; Cd 30 G>C; IVS 1 , nt 2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5 G>A; IVS 1 , nt 5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt 844 OG; IVS 2,nt 844 OA βS (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (Cd 126) T>G ; wherein Cd: codon ; IVS: intron; Fr: frameshift ; nt: nucleotide. Figure 2 shows the localization of the above mentioned mutations at level of β globin gene. Therefore, it is an object of the present invention oligonucleotide probes able to hybridize to the target sequence belonging to the β globin gene that may comprise the sequences:
AG A AC TCT GGG TCC AAG (SEQ ID No 1);
AGA ACC TCT AGG TCC AA (SEQ ID No 2); TC TGC CTA TTG GTC TAT TTT (SEQ ID No 3);
CT GCC TAT TAG TCT ATT TTC (SEQ ID No 4);
AT ACC AACCTG CCC AG (SEQ ID No 5);
CT GGG CAG ATT GGT AT (SEQ ID No 6);
CC TTG ATA CCA ACC TG (SEQ ID No 7); AG GTT GGC ATC AAG GT (SEQ ID No 8) ;
AAC TTC AGG GTG AGT CTA T (SEQ ID No 9);
AAC TTC AGG ATG AGT CTA T (SEQ ID No 10);
CA ATC CAG CTA CCA TTC (SEQ ID No 11 );
GA ATG GTA CCT GGA TTG (SEQ ID No 12); CTT CTC CTC AGG AGT C (SEQ ID No 13);
TT CTC CTC GAG TCA GGT (SEQ ID No 14);
TGA CTC CTG GGA GAA G (SEQ ID No 15);
GAC TCC TGT GGA GAA G (SEQ ID No 16);
CTG ACT CCT AAG GAG AAG (SEQ ID No 17); TG AGG AGA AGT CTG CC (SEQ I D No 18);
AG GGC AGA CCT CCT CA (SEQ ID No 19);
AGG AGA AGG TCT GCC G (SEQ ID No 20);
AC CTC ACC CTG TGG AG (SEQ ID No 21 ); GC TCC ACA AGG TGA GGT (SEQ ID No 22) ;
GT GGA GCC ACA CCC TA (SEQ ID No 23);
TA GGG TGT AGC TCC AC (SEQ ID No 24);
AC CCT AGG TTG TGG CT (SEQ ID No 25); AC CCT AGG ATG TGG CT (SEQ ID No 26) ;
AA CCC TAG GGT GTG GCT (SEQ ID No 27) ;
AA CCC TAG CGT GTG GCT (SEQ ID No 28) ;
AA CCC TAG AGT GTG GCT (SEQ ID No 29) ;
CA ACC CTA TGG TGT GG (SEQ ID No 30); CT GGG CAT AAA AGT CAG (SEQ ID No 31 );
T GAC TTT TTT GCC CAG (SEQ ID No 32);
TG ACT TTT GTG CCC AG (SEQ ID No 33);
CT GGG CAC GTT GGT A (SEQ ID No 34);
TG GGC AGG ATG GTA TC (SEQ ID No 35); GG GCA GGG TGG TAT C (SEQ ID No 36);
C AGG TTG ATA TCA AGG (SEQ ID No 37);
GC AGG TTG CTA TCA AG (SEQ ID No 38);
C AGG TTG TTA TCA AGG (SEQ ID No 39) ;
TT TGA GTC CTT TGG GGA (SEQ ID No 40); CTT TGA GTC TTT GGG GA (SEQ ID No 41 );
GG CCT GGC TCA CCT G (SEQ ID No 42);
CC AGG TGA CCA GGC C (SEQ ID No 43);
AG GAG AAG GTC TGC CG (SEQ ID No 44);
AT CTT CCT CCC ACA GC (SEQ ID No 45); GC TGT GGC AGG AAG AT (SEQ I D No 46);
GC TGT GGT AGG AAG AT (SEQ ID No 47) ;
CCA CCA GTG CAG GCT (SEQ ID No 48);
GC CTG CCC TGG TGG (SEQ ID No 49); or the complementary sequences thereof or the sequences wherein the T is replaced with U. In particular, the target sequence belonging to the β-globin gene may comprise at least one mutated sequence selected from the group consisting of:
Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92
OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8
(-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5
G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; β S (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (cd126) T>G; and/or the corresponding unmutated sequence. The oligonucleotide probes according to the present invention can be chemically modified and/or labelled, said probes being modified at the 5' end with an aminic group or with a poli-T sequence. This type of chemical modification is significant to allow the possibile immobilization of said oligonucleotide probes to a solid support. In fact, the aminomodified or differently derivatized oligonucleotide probes can be anchored to a solid support, preferably to an opportunely activated membrane, negatively or positively charged. In the case of 5'aminomodified probes the membrane is negatively charged and is preferably activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In the specific case of the 5' poli-T derivatization a positively charged membrane as a solid support, would be necessary. Therefore, it is a further object of the present invention an opportunely activated solid support, comprising at least one of the above oligonucleotide probes anchored thereon. In particular, said solid support can be an opportunely activated membrane with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The oligonucleotide probes can be alternatively labelled with at least one fluorochrome or a radioactive isotope at the 3' and/or 5' ends of the oligonucleotide sequence. The fluorochrome may be an emission and/or excitation fluorochrome, preferably it is fluorescein, for the detection, for example, by PCR Real time. Further object of the present invention is the use of the oligonucleotide sequences according to the present invention, for the diagnosis and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects. According to another aspect the present invention concerns a method for the detection and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects, comprising the following steps: a) DNA extraction from the biological sample (any organic tissue: blood, amniotic liquid, chorial villi liquid) under examination; b) PCR amplification of β-globin gene containing the target sequence through the use of a set of flanking primer of the β-globin gene; c) detection of the mutated target sequence and/or the corresponding unmutated sequence amplified in step b) through the use of oligonucleotide probes according to the present invention able to hybridize themselves to said target sequence belonging to the β-globin gene. Specifically, the set of primer of step b) of the above method may comprise at least one pair of primer selected from the group of oligonucleotide sequences:
AGA GAT ATA TCT TAG AGG GAG (SEQ ID No 50); GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51 ); CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52);
TCA TTC GTC TGT TTC CCA TTC TAA AC (SEQ ID No 53); TGC ATA TTC ATA ATC TCC CTA CTT T (SEQ ID No 54); CAC TGA CCT CCC ACA TTC CC (SEQ ID No 55); or the complementary sequences thereof. The mutated target sequence of step c) of the above method may comprise a sequence with at least one mutation, said mutation being selected from the group consisting of:
Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92 OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8 (-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5 G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; βS (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (Cd 126) T>G. According to a preferred embodiment of the present invention the oligonucleotide probes used in step c) of the method according to the present invention can be anchored to a solid support. Said solid support may be a negatively or positively charged membrane, opportunely activated. Said activation can be carried out with 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide in the case of a negatively charged membrane to be employed as a solid support. It is an object of the present invention a diagnostic kit for the detection and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects, which may comprise: a) a set of primer for the PCR amplification of β-globin gene containing the target sequence, which may comprise at least two among the following oligonucleotide sequences: AGA GAT ATATCT TAG AGG GAG (SEQ ID No 50);
GTA CGG CTG TCATCA CTTAGA CCT (SEQ ID No 51);
CAA CTT CAT CCA CGTTCA CC (SEQ ID No 52);
TCATTC GTC TGT TTC CCATTC TAAAC (SEQ ID No 53); TGC ATATTC ATAATC TCC CTA CTT T (SEQ ID No 54);
CAC TGA CCT CCC ACATTC CC (SEQ ID No 55); or the complementary sequence thereof; b) oligonucleotide probes according to the present invention able to hybridize to said target sequence. In particular, the mutations detected and identified by the diagnostic kit according to the present invention may be selected from the group consisting in:
Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt
1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92 OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8
(-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5
G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130
G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; βS
(cd 6) A>T; βC (cd6) G>A; Hb Neapolis (cd126) T>G. According to a particular embodiment the oligonucleotide probes of the diagnostic kit according to the present invention can be anchored to a solid support. Said solid support can be a positively or negatively charged membrane, opportunely activated. Said activation can be carried out with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the case of a negatively charged membrane to be employed as a solid support. Further object of the present invention are primers for the PCR amplification of the β-globin gene that may comprise at least one of the sequences: AGA GAT ATA TCT TAG AGG GAG (SEQ ID No 50);
GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51 );
CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52);
TCA TTC GTC TGT TTC CCA TTC TAA AC (SEQ ID No 53);
TGC ATA TTC ATA ATC TCC CTA CTT T (SEQ ID No 54); CAC TGA CCT CCC ACA TTC CC (SEQ ID No 55); or the complementary sequence thereof. Said primers can be previously labelled with biotin at the 5' end. Finally, object of the present invention are antisense oligonucleotides that may comprise at least one of the sequences:
AG A AC TCT GGG TCC AAG (SEQ ID No 1 );
AGA ACC TCT AGG TCC AA (SEQ ID No 2); TC TGC CTA TTG GTC TAT TTT (SEQ ID No 3);
CT GCC TAT TAG TCT ATT TTC (SEQ ID No 4);
AT ACC AACCTG CCC AG (SEQ ID No 5);
CT GGG CAG ATT GGT AT (SEQ ID No 6);
CC TTG ATA CCA ACC TG (SEQ ID No 7); AG GTT GGC ATC AAG GT (SEQ ID No 8) ;
AAC TTC AGG GTG AGT CTA T (SEQ ID No 9);
AAC TTC AGG ATG AGT CTA T (SEQ ID No 10);
CA ATC CAG CTA CCA TTC (SEQ ID No 11 );
GA ATG GTA CCT GGA TTG (SEQ ID No 12); CTT CTC CTC AGG AGT C (SEQ ID No 13);
TT CTC CTC GAG TCA GGT(SEQ ID No 14);
TGA CTC CTG GGA GAA G (SEQ ID No 15);
GAC TCC TGT GGA GAA G (SEQ ID No 16);
CTG ACT CCT AAG GAG AAG (SEQ ID No 17); TG AGG AGA AGT CTG CC (SEQ I D No 18);
AG GGC AGA CCT CCT CA (SEQ ID No 19);
AGG AGA AGG TCT GCC G (SEQ ID No 20);
AC CTC ACC CTG TGG AG (SEQ ID No 21 );
GC TCC ACA AGG TGA GGT (SEQ ID No 22) ; GT GGA GCC ACA CCC TA (SEQ ID No 23);
TA GGG TGT AGC TCC AC (SEQ ID No 24);
AC CCT AGG TTG TGG CT (SEQ ID No 25);
AC CCT AGG ATG TGG CT (SEQ ID No 26) ;
AA CCC TAG GGT GTG GCT (SEQ ID No 27) ; AA CCC TAG CGT GTG GCT (SEQ ID No 28) ;
AA CCC TAG AGT GTG GCT (SEQ ID No 29) ;
CA ACC CTA TGG TGT GG (SEQ ID No 30);
CT GGG CAT AAA AGT CAG (SEQ ID No 31 );
T GAC TTT TTT GCC CAG (SEQ ID No 32); TG ACT TTT GTG CCC AG (SEQ ID No 33);
CT GGG CAC GTT GGT A (SEQ ID No 34);
TG GGC AGG ATG GTA TC (SEQ ID No 35);
GG GCA GGG TGG TAT C (SEQ ID No 36); C AGG TTG ATA TCA AGG (SEQ ID No 37);
GC AGG TTG CTA TCA AG (SEQ ID No 38);
C AGG TTG TTA TCA AGG (SEQ ID No 39) ;
TT TGA GTC CTT TGG GGA (SEQ ID No 40); CTT TGA GTC TTT GGG GA (SEQ ID No 41 );
GG CCT GGC TCA CCT G (SEQ ID No 42);
CC AGG TGA CCA GGC C (SEQ ID No 43);
AG GAG AAG GTC TGC CG (SEQ ID No 44);
AT CTT CCT CCC ACA GC (SEQ ID No 45); GC TGT GGC AGG AAG AT (SEQ ID No 46);
GC TGT GGT AGG AAG AT (SEQ ID No 47) ;
CCA CCA GTG CAG GCT (SEQ ID No 48);
GC CTG CCC TGG TGG (SEQ ID No 49); or the complementary sequence thereof or the sequences wherein the T is replaced with U. It is a further object of the present invention the use of the antisense oligonucleotides both for the manufacture of a medicament for the treatment of β-thalassemia and for diagnosis and identification of the β-globin gene mutations associated with β-thalassemia. The present invention will be now described with reference to exemplifying, but not limiting, preferred embodiments thereof, with a particular reference to the figures of the enclosed drawings, wherein: Figure 1 shows the detection scheme typical of a generic
Reverse Dot Blot (RDB) assay, wherein, after the binding of the conjugated streptavidin with alkaline phosphatase (AP) to the biotin, a colorimetric reaction and the subsequent formation of an insoluble precipitate in presence of two chromogens take place; Figure 2 shows the localization of the gene mutations associated with β-thalassemia, indicated with an abbreviation, at level of the β-globin gene locus; EX: exon; IVS: intron; Cd: codon; nt: nucleotide; Figure 3, in panels A, B, C, D, E shows the phenotypes of the different forms of β°-thalassemia and β+-thalassemia. In particular, panel A shows the phenotype of those subjects who are not carrier of β- thalassemia; panel B shows the phenotype of those subjects with β°- thalassemia; panel C shows the phenotype of those subjects with severe β+-thalassemia; panel D shows the phenotype of those subjects with minor β+-thalassemia; panel E shows the phenotype of those subjects with silent β+-thalassemia. EXAMPLE 1 : Reverse Dot Blot experiment using the oligonucleotide probes for the detection and the identification of gene mutations associated with β-thalassemia MATERIALS AND METHODS Reverse Dot Blot (RDB) was carried out on 10.000 DNA samples from β-thalassemia healthy carrier and from normal subjects. The results achieved during the study carried out by the authors of the present invention were showed in Table 1. Table 1 shows the incidence frequency and the genetic mutation type associated with β- thalassemia, analysed in a group of patients belonging to the Sicilian population (10.000 chromosomes). Table 1
Figure imgf000013_0001
Figure imgf000014_0001
Further, over 2000 β-thalassemia pre-birth diagnosis were carried out. All the results achieved by the RDB technique were confirmed by other molecular biology systems such as: restriction endonucleases analysis, ARMS, gene sequencing; all the data were confirmed. Amplification conditions The DNA genomic region to be analysed for the detectable mutations by RDB technique, employing the oligonucleotide sequences according to the present invention as probes, was analysed by PCR with the use of the Taq polymerase enzyme. During the course of this PCR reaction a deoxynucleotide (dUTP) conjugated with a biotin molecule was inserted into the DNA of new generation or 5' biotinilated primer were used. Amplified DNA labelling can be carried out through two way: 1) using the biotin-16-dUTP base during the course of PCR, which links randomly in place of thymine; 2) using biotinilated modified primer at 5' (primer-5'-biotin-sequence). The PCR amplification conditions DNA samples to be undergone RDB analysis were reported in Table 2. Table 2
Figure imgf000015_0001
The oligonucleotide sequences of the primer according to the present invention that can be used for the amplification of the β-globin gene are the following: 1) AGAGAT ATA TCT TAG AGG GAG (SEQ ID No 50) 2) GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51) 3) CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52) 4) TCA TTC GTC TGT TTC CCA TTC TAA AC (SEQ ID No 53) 5) TGC ATA TTC ATA ATC TCC CTA CTT T (SEQ ID No 54) 6) CAC TGA CCT CCC ACA TTC CC (SEQ ID No 55) Subsequently, the biotinilated PCR product (biotinilation carried out by using previously biotinilated primers or by adding biotin) was hybridized (hybridization in liquid phase) with the oligonucleotide probes anchored to the membrane. Membrane activation The nylon membrane was soaked with a 10% EDC solution and was shaken at room temperature for 15 minutes. Then the membrane was rinsed with dH20 and dried at room temperature. Negatively charged pre-activate membrane can be used for the anchorage of the 5' NH2-modified probes or positively charged membrane can be used for poli-T 5' modified probes. Membrane preparation The nylon membrane was separated in rectangular sections ("strips" of about 1 ,5 X 6 cm). 1 ,5 μl of each oligonucleotide probe were laid down on the membrane being careful to place the oligonucleotides corresponding to the normal sequences on the right, and those corresponding to the mutated sequences on the left of each strip. The oligonucleotide sequences of the 5' aminomodified probes that hybridize to the unmutated sequences were shown in Table 3.
Table 3
Figure imgf000016_0001
The oligonucleotide sequences of the 5' aminomodified probes that hybridize to the mutated sequences were shown in Table 4. Table 4
Figure imgf000017_0001
Subsequently, the dry membrane was inactivated by soaking with 0,1 M NaOH solution and was shaken for 10 minutes. Therefore, the NaOH solution was removed and the membrane was rinsed with dH 0 for two times running. Membranes hybridization The membrane was cut in such a way to obtain individual strips that were inserted in their turn into single 15 ml screw tubes. 5 ml of "hybridization solution" (2X SSC + 0,1% SDS) were added and tubes were incubated at 45°C for 10 minutes under shaking. 0,5 ml of hybridization solution were added into an 1 ,5 ml eppendorf tube for each sample to be analyzed and subsequently 22,5 μl of amplified DNA were added for each tube. DNA denaturation was carried out maintaining the eppendorf tubes at a temperature of 100°C for 10 minutes. The DNA thus denaturated was transferred into the 15 ml tube containing the membrane, being careful not to pour the content directly onto the membrane. Tubes were incubated at a temperature of 45°C for 60 minutes and maintained under shaking. Membrane washing The membranes were extracted from 15 ml tubes, and collected in a becker containing about 200 ml of hybridization solution at a temperature of 45°C and were maintained under shaking for 20 minutes. At the end of the hybridization reaction, the membrane with the oligonucleotide sequences, with the DNA amplificates eventually hybridized thereto, was contacted with a solution containing streptavidin molecules conjugated with alkaline phosphatase or with peroxidase. A mixture containing 7,5 μl of streptavidin-AP (1 U/μl) in 20 ml of hybridization solution, was prepared. Strips were incubated in the streptavidin solution at room temperature and shaken for 45 minutes In case wherein the alternative system of streptavidin conjugated with peroxidase was used, 1 μl/10 ml of streptavidin- horseradish peroxidase conjugate (1 mg/ml) were employed. The streptavidin-AP solution was removed; therefore 50 ml of the hybridization solution were added and the membranes were maintained under shaking for 10 minutes at room temperature. This last operation was repeated another time. Strips were incubated with 50 ml of Genius Buffer at room temperature and maintained under shaking for 5 minutes. Also this operation was repeated again. In case wherein the streptavidin-peroxidase conjugate was used, the incubation has to be carried out twice for 5 minutes in 100 mmol/L sodium citrate pH 5. Color development The streptavidin binds the amplified DNA incorporated biotin, while alcaline phosphatase will react with a chromogen containing a phosphate mixture (NBT and BICP) catalyzing the phosphate transfer reaction from BICP to NBT thus resulting in the formation of a blu insoluble precipitate at level of the probe. In case wherein streptavidin-peroxidase conjugate is used, the chromogen is a 0,1 mg/ml solution of 3\3',5',5',tetramethylbenzidine, 000,3% H202 in 100 mmol/L sodium citrate pH 5. The unmutated sequence complementary to the normal sequences, as well as the oligonucleotide sequences complementary to the mutated sequences (Table 4), were anchored on the strips (Table 3), in such a way to ascertain if the patient under examination is heterozygote or homozygote for the searched gene mutation. The staining solution was prepared by adding 45 μl of NBT and 35 μl of BCIP to the aliquotes of 10 ml of Genius Buffer. 10 ml of staining solution were prepared for every 6 strips, because such solution must cover totally the strips, that must not overlap each other . In case wherein streptavidin-peroxidase conjugate was used the chromogen is a 0,1 mg/ml solution of 3', 3', 5', 5', tetramethylbenzidine, 000,3% H202, in 100 mmol/L sodium citrate pH 5. The strips were incubated in the dark at room temperature until stained spots appear, for about 15-45 minutes. Finally, were washed in dH20 and dried at room temperature. CONCLUSIONS The Reverse Dot Blot was designed and tested to the laboratory of pre-birth diagnosis of thalassemia which is centralized to the
Ematologia II U.O. of the Hospital Vincenzo Cervello in Palermo. The
Ematologia II U.O. is the main centre of the Sicily Region for the thalassemia treatment and pre-birth diagnosis. The experimentation was carried out by using the samples of normal subjects and healthy carriers of β-thalassemia with the phenotypes as described in figure 3, panels A, B, C, D, E. The panel A shows the phenotype of subjects who were not carriers of β-thalassemia. The MCV value is between 80 and 90 fl, while HbA2 is less than 3,5%. The panel B shows the phenotype of subjects with β°- thalassemia, mainly characterized in that the MCV value is very low and HbA2 is very high. The panel C shows the phenotype of subjects with severe β+- thalassemia. It is notable that the MCV value was increased in comparison to the β°-thalassemia while HbA2 value is less than 5%. The panel D shows the phenotype of subjects with minor β+- thalassemia. The MCV value was increased in comparison to the severe β+-thalassemia (around 70 fl) while HbA2 value is about 4%. The panel E shows the phenotype of subjects with silent β+- thalassemia. The MCV value was normal (> 80 fl) and the HbA2 value may range between 3,3% to 4%. During the experimental stage, all the results were confirmed by biologic molecular techniques that are already used at U.O. using negative and positive control samples. In those cases wherein the restriction enzyme able to recognize one of the mutation object of the experimentation was disposable this system was employed; in the absence of the restriction enzyme the ARMS techniques or the direct gene sequencing were used. During the experimentation the designed sequences were designed since the results were not due to the cross-hybridization (false positives) or to the absence of signal. 2000 β-thalassemia positive samples and 100 normal samples were employed for the experimentation. The Reverse Dot Blot system through the use of the sequences object of the present invention is able to extabilish the genotype of the 95% mutations present in the Mediterranean area. The aforesaid Reverse Dot Blot is a particularly versatile system, since the oligoprobes work at the same hybridization and washing temperature. Therefore, it is possible to manufacture kits or membrane with the probes of the present invention to be correlated to the mutations present inside a certain population (4) or to the phenotype of the subject under examination. Therefore such a system is suitable for the molecular screening of those subject who carry β-thalassemia and for the pre-birth diagnosis and may be adjusted for the analysis of other globin genes (5). BIBLIOGRAPHY (1) Giambona, P. Lo Gioco, M. Marino, I. Abate, R. Di Marzo, M. Renda, F. Di Trapani, F. Messana, S. Siciliano, P. Rigano, F.F. Chehab, H.H. Kazazian, A. Maggio (1995).Hum. Genet,. 95:526-530. (2) Chehab FF, Wall J. (1992). Human genetics 89: 163-168
(3) Maggio A, Giambona A, CaiSP, Wall J, Kan YW, Chehab FF (1993).Application to prenatal diagnosis in Sicily. Blood 81 : 239-242.
(4) Sutcharitchan P, Saiki R, Husman THJ, McKie V, Erlich V, Embury SH. (1995). Blood, 86, #4 1580-1585. (5) E. Foglietta, I. Bianco, A. Maggio, A. Giambona. (2003). American Journal of Hematology 94, #3 pg 123-127.

Claims

1. Oligonucleotide probes able to hybridize to a target sequence belonging to the β-globin gene comprising the sequences:
AG A AC TCT GGG TCC AAG (SEQ ID No 1 ); AGA ACC TCT AGG TCC AA (SEQ ID No 2);
TC TGC CTA TTG GTC TAT TTT (SEQ ID No 3);
CT GCC TAT TAG TCT ATT TTC (SEQ ID No 4);
AT ACC AACCTG CCC AG (SEQ ID No 5);
CT GGG CAG ATT GGT AT (SEQ ID No 6); CC TTG ATA CCA ACC TG (SEQ ID No 7);
AG GTT GGC ATC AAG GT (SEQ ID No 8) ;
AAC TTC AGG GTG AGT CTA T (SEQ ID No 9);
AAC TTC AGG ATG AGT CTA T (SEQ ID No 10);
CA ATC CAG CTA CCA TTC (SEQ ID No 11 ); GA ATG GTA CCT GGA TTG (SEQ ID No 12);
CTT CTC CTC AGG AGT C (SEQ ID No 13);
TT CTC CTC GAG TCA GGT (SEQ ID No 14);
TGA CTC CTG GGA GAA G (SEQ ID No 15);
GAC TCC TGT GGA GAA G (SEQ ID No 16); CTG ACT CCT AAG GAG AAG (SEQ ID No 17);
TG AGG AGA AGT CTG CC (SEQ ID No 18);
AG GGC AGA CCT CCT CA (SEQ ID No 19);
AGG AGA AGG TCT GCC G (SEQ ID No 20);
AC CTC ACC CTG TGG AG (SEQ ID No 21 ); GC TCC ACA AGG TGA GGT (SEQ ID No 22) ;
GT GGA GCC ACA CCC TA (SEQ ID No 23);
TA GGG TGT AGC TCC AC (SEQ ID No 24);
AC CCT AGG TTG TGG CT (SEQ ID No 25);
AC CCT AGG ATG TGG CT (SEQ ID No 26) ; AA CCC TAG GGT GTG GCT (SEQ ID No 27) ;
AA CCC TAG CGT GTG GCT (SEQ ID No 28) ;
AA CCC TAG AGT GTG GCT (SEQ ID No 29) ;
CA ACC CTA TGG TGT GG (SEQ ID No 30);
CT GGG CAT AAA AGT CAG (SEQ ID No 31 ); T GAC TTT TTT GCC CAG (SEQ ID No 32);
TG ACT TTT GTG CCC AG (SEQ ID No 33);
CT GGG CAC GTT GGT A (SEQ ID No 34);
TG GGC AGG ATG GTA TC (SEQ ID No 35); GG GCA GGG TGG TAT C (SEQ ID No 36);
C AGG TTG ATA TCA AGG (SEQ ID No 37);
GC AGG TTG CTA TCA AG (SEQ ID No 38);
C AGG TTG TTA TCA AGG (SEQ ID No 39) ; TT TGA GTC CTT TGG GGA (SEQ ID No 40);
CTT TGA GTC TTT GGG GA (SEQ ID No 41 );
GG CCT GGC TCA CCT G (SEQ ID No 42);
CC AGG TGA CCA GGC C (SEQ ID No 43);
AG GAG AAG GTC TGC CG (SEQ ID No 44); AT CTT CCT CCC ACA GC (SEQ I D No 45);
GC TGT GGC AGG AAG AT (SEQ ID No 46);
GC TGT GGT AGG AAG AT (SEQ ID No 47) ;
CCA CCA GTG CAG GCT (SEQ ID No 48);
GC CTG CCC TGG TGG (SEQ ID No 49); or the complementary sequences thereof or the sequences wherein the T is replaced with U.
2. Oligonucleotide probes according to claim 1 , wherein said target sequence belonging to the β-globin gene comprising at least one mutated sequence selected from the group consisting in: Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92 OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8 (-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5 G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; β S (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (cd126) T>G; and/or the corresponding unmutated sequence.
3. Oligonucleotide probes according to anyone of the claims 1 and 2, wherein said probes are chemically modified and/or labelled.
4. Oligonucleotide probes according to anyone of the preceding claims, wherein said probes are modified at the 5' end with an aminic group or with a poli-T sequence.
5. Oligonucleotide probes according to claims 3 or 4, wherein said oligonucleotide were anchored to a solid support.
6. Oligonucleotide probes according to anyone of the claims 1 to 3, wherein said probes are labelled with at least one fluorochrome or a radioactive isotope at the 3' and/or 5' ends of the oligonucleotide sequence.
7. Oligonucleotide probes according to claim 6, wherein the fluorochrome is an emission and/or excitation fluorochrome.
8. Oligonucleotide probes according to claims 6 or 7, wherein the emission fluorochrome is fluorescein .
9. Solid support comprising at least one probe, characterized in that said at least one probe is an oligonucleotide probe according to anyone of the claims 1 to 4.
10. Solid support according to claim 9, wherein said solid support is a positively or negatively charged membrane.
11. Solid support according to claim 10, wherein said negatively charged membrane is activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
12. Use of at least one of the oligonucleotide sequences as defined in claims 1 to 8, for the diagnosis and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects.
13. Use according to claim 12, wherein the diagnosis is carried out by a technique selected from the group consisting of PCR, Reverse Dot Blot (RBD), in situ hybridization, Multiple Tissue Array (MTA).
14. Method for the detection and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects, comprising the following steps: a) DNA extraction from the biological sample under examination; b) PCR amplification of the β-globin gene containing the target sequence through the use of a set of flanking primer of the β-globin gene; c) detection of the mutated target sequence and/or the corresponding unmutated sequence amplified in step b) through the use of oligonucleotide probes as defined in claims 1 to 8 able to hybridize themselves to said target sequence belonging to the β-globin gene.
15. Method according to claim 14, wherein the set of primer of step b) comprise at least one pair of primer selected between the oligonucleotide sequences:
AGA GAT ATA TCT TAG AGG GAG (SEQ ID No 50);
GTA CGG CTG TCA TCA CTT AGA CCT (SEQ ID No 51 ); CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52);
TCA TTC GTC TGT TTC CCA TTC TAA AC (SEQ ID No 53);
TGC ATA TTC ATA ATC TCC CTA CTT T (SEQ ID No 54);
CAC TGA CCT CCC ACA TTC CC (SEQ ID No 55); or the complementary sequences thereof.
16. Method according to anyone of the claims 14 and 15, wherein the mutated target sequence of step c) comprises a sequence with at least one mutation selected from the group consisting of: Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92 OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8 (-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5 G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; βS (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (Cd 126) T>G.
17. Method according to anyone of the claims 14 to 16, wherein the oligonucleotide probes used in the step c) were anchored to a solid support.
18. Method according to claim 17, wherein said solid support is an activated membrane.
19. Method according to anyone of the claims 17 and 18, wherein said membrane is activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
20. Diagnostic kit for the detection and the identification of the β-globin gene mutations associated with β-thalassemia, in heterozygote and homozygote subjects, comprising: a) a set of primer for the PCR amplification of β-globin gene containing the target sequence; b) oligonucleotide probes as defined in anyone of the claims 1 to 8 able to hybridize to said target sequence.
21. Diagnostic kit according to claim 20, wherein said mutations are selected from the group consisting in:
Cd 39 OT; IVS 1 , nt 110 G>A; IVS 1 , nt 1 G>A; IVS 1 ,nt 6T>C; IVS 2, nt 1 G>A; IVS 2, nt 745 OG; Fr Cd 6 -A; -87 OG; -87 OT; -101 OT; -92 OT; -88 OA; -88 OT; -86 OA; -30 T>A; -30 T>C; Fr Cd5 -CT; Fr Cd 8 (-AA); Cd 8/9 (+G); Cd30 G>C; IVS 1 , nt2 T>A; IVS 1 ,nt 2 T>G; IVS 1 ,nt 5 G>A; IVS1 , nt5 G>C ; IVS 1 ,nt 5 G>T; IVS 2,nt 116 T>G; IVS 1 ,nt 130 G>C; Fr Cd44 -C; Fr Cd 76 -C; IVS 2, nt844 OG; IVS 2,nt 844 OA; βS (cd 6) A>T; βC (cd6) G>A; Hb Neapolis (cd126) T>G.
22. Diagnostic kit according to anyone of the claims 20 and 21 , wherein the set of primer comprises at least one pair of primer selected from the oligonucleotide sequences: AGA GATATATCTTAG AGG GAG (SEQ ID No 50);
GTA CGG CTG TCA TCA CTTAGA CCT (SEQ ID No 51);
CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52);
TCATTC GTC TGT TTC CCATTC TAAAC (SEQ ID No 53); TGC ATATTC ATAATC TCC CTA CTT T (SEQ ID No 54);
CAC TGA CCT CCC ACATTC CC (SEQ ID No 55); or the complementary sequence thereof;
23. Diagnostic kit according to anyone of the claims 20 to 22, wherein said oligonucleotide probes are anchored to a solid support.
24. Diagnostic kit according to claim 23, wherein said solid support is an activated membrane.
25. Diagnostic kit according to anyone of the claims 23 and 24, wherein said membrane is activated with 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide.
26. Primers for the PCR amplification of the β-globin gene comprising at least one of the sequences:
AGA GAT ATATCT TAG AGG GAG (SEQ ID No 50);
GTA CGG CTG TCATCA CTTAGA CCT (SEQ ID No 51);
CAA CTT CAT CCA CGT TCA CC (SEQ ID No 52); TCATTC GTC TGT TTC CCATTC TAAAC (SEQ ID No 53);
TGC ATATTC ATAATC TCC CTA CTT T (SEQ ID No 54);
CAC TGA CCT CCC ACATTC CC (SEQ ID No 55); or the complementary sequences thereof.
27. Primers according to claim 26, wherein said primer are labelled with biotin at the 5' end.
28. Antisense oligonucleotides comprising at least one of the sequences:
AG A AC TCT GGG TCC AAG (SEQ ID No 1 );
AGA ACC TCT AGG TCC AA (SEQ ID No 2); TC TGC CTA TTG GTC TAT TTT (SEQ ID No 3);
CT GCC TAT TAG TCT ATT TTC (SEQ ID No 4);
AT ACC AACCTG CCC AG (SEQ ID No 5);
CT GGG CAG ATT GGT AT (SEQ ID No 6);
CC TTG ATA CCA ACC TG (SEQ ID No 7); AG GTT GGC ATC AAG GT (SEQ ID No 8) ;
AAC TTC AGG GTG AGT CTA T (SEQ ID No 9);
AAC TTC AGG ATG AGT CTA T (SEQ ID No 10);
CA ATC CAG CTA CCA TTC (SEQ ID No 11 ); GA ATG GTA CCT GGA TTG (SEQ ID No 12);
CTT CTC CTC AGG AGT C (SEQ ID No 13);
TT CTC CTC GAG TCA GGT (SEQ ID No 14);
TGA CTC CTG GGA GAA G (SEQ ID No 15); GAC TCC TGT GGA GAA G (SEQ ID No 16);
CTG ACT CCT AAG GAG AAG (SEQ ID No 17);
TG AGG AGA AGT CTG CC (SEQ ID No 18);
AG GGC AGA CCT CCT CA (SEQ ID No 19);
AGG AGA AGG TCT GCC G (SEQ ID No 20); AC CTC ACC CTG TGG AG (SEQ I D No 21 );
GC TCC ACA AGG TGA GGT (SEQ ID No 22) ;
GT GGA GCC ACA CCC TA (SEQ ID No 23);
TA GGG TGT AGC TCC AC (SEQ ID No 24);
AC CCT AGG TTG TGG CT (SEQ ID No 25); AC CCT AGG ATG TGG CT (SEQ ID No 26) ;
AA CCC TAG GGT GTG GCT (SEQ ID No 27) ;
AA CCC TAG CGT GTG GCT (SEQ ID No 28) ;
AA CCC TAG AGT GTG GCT (SEQ ID No 29) ;
CA ACC CTA TGG TGT GG (SEQ ID No 30); CT GGG CAT AAA AGT CAG (SEQ I D No 31 );
T GAC TTT TTT GCC CAG (SEQ ID No 32);
TG ACT TTT GTG CCC AG (SEQ ID No 33);
CT GGG CAC GTT GGT A (SEQ ID No 34);
TG GGC AGG ATG GTA TC (SEQ ID No 35); GG GCA GGG TGG TAT C (SEQ ID No 36);
C AGG TTG ATA TCA AGG (SEQ ID No 37);
GC AGG TTG CTA TCA AG (SEQ ID No 38);
C AGG TTG TTA TCA AGG (SEQ ID No 39) ;
TT TGA GTC CTT TGG GGA (SEQ ID No 40); CTT TGA GTC TTT GGG GA (SEQ ID No 41 );
GG CCT GGC TCA CCT G (SEQ ID No 42);
CC AGG TGA CCA GGC C (SEQ ID No 43);
AG GAG AAG GTC TGC CG (SEQ ID No 44);
AT CTT CCT CCC ACA GC (SEQ ID No 45); GC TGT GGC AGG AAG AT (SEQ ID No 46);
GC TGT GGT AGG AAG AT (SEQ ID No 47) ;
CCA CCA GTG CAG GCT (SEQ ID No 48);
GC CTG CCC TGG TGG (SEQ ID No 49); or the complementary sequences thereof or the sequences wherein the T is replaced with U.
29. Use of the antisense oligonucleotides according to claim 28, for the preparation of a medicament for the treatment of β-thalassemia.
30. Use of the antisense oligonucleotides according to claim 28, for the diagnosis and the identification of the β-globin gene mutations associated with β-thalassemia.
PCT/IT2005/000066 2004-02-13 2005-02-11 OLIGONUCLEOTIDE PROBES FOR DIAGNOSIS AND IDENTIFICATION OF THE DIFFERENT FORMS OF β-THALASSEMIA, METHODS AND DIAGNOSTIC KIT THEREOF Ceased WO2005078132A2 (en)

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