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  • 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
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• Thalassemia is an inherited genetic, i.e. autosomal recessive blood disorder.
• the genetic defect which can be a mutation or a deletion, typically results in a reduced rate of synthesis of one of the globin chains of hemoglobin, or in no synthesis of these chains.
• abnormal hemoglobin molecules are formed, which lead to anemia, i.e. the characteristic symptom of all thalassemia forms.
• thalassemias are thus related to quantitative problems of a reduced number of globins synthesized, often via mutations or modifications in regulatory genes or regions, whereas the other predominant anemic disorder sickle-cell anemia is caused by the qualitative problem of the synthesis of mal- functioning globins.
• the genetic situation, or the mutations leading to thalassemia minor or beta thalassemia trait, are typically described as ⁇ / ⁇ or ⁇ °/ ⁇ . Due to the autosomal recessive inheritance of the disease beta thalassemia minor carriers, however, pose a major threat to public health since in subsequent generations combinations of recessive traits may lead to more severe forms of the disease.
• beta globin gene cluster Genetics, 11, 51).
• a point mutation in codon 26 of the beta globin gene can induce alternative splicing which results in decreased beta globin E chains, leading to hypochromic microcytosis and minimal to severe anemia.
• Sherva et al. discovered 50 single nucleotide polymorphisms associated with this specific thalassemia form, which were mostly functionally linked to a regulatory region centromeric of the beta globin gene cluster.
• beta thalassemia can be found in populations in the Mediterranean region, in North Africa, West Asia and South Asia, which show the world's highest concentration of carriers. For example, in India, the carrier rate of beta thalassemia is assumed to be 3-17%.
• beta thalassemia may become a very serious problem in the next decades, which may, inter alia, burden the world's blood bank supplies and the health system in general.
• SEQ ID NO: 1 except for a single polymorphic change at position 501, where wildtype nucleotide T is replaced by indicator nucleotide C
• SEQ ID NO: 2 except for a single polymorphic change at position 501, where wildtype nucleotide T is replaced by indicator nucleotide C
• an indicator nucleotide as defined herein above is indicative of the presence of beta thalassemia minor.
• the above mentioned sample may be a mixture of tissues, organs, cells and/or fragments thereof, or a tissue or organ specific sample, such as a tissue biopsy from vaginal tissue, tongue, pancreas, liver, spleen, ovary, muscle, joint tissue, neural tissue, gastrointestinal tissue, tumor tissue, or a body fluid, blood, serum, saliva, or urine. Particularly preferred is blood.
• the method as mentioned herein above comprises the determination of the nucleotide sequence and/or molecular structure present at polymorphic sites of SEQ ID NO: 8 and SEQ ID NO: 9 and the detection of a DNAse hypersensitivity site in the genomic vicinity of SEQ ID NO: 8 and/or SEQ ID NO: 9, wherein the presence of an indicator nucleotide as defined herein above and the presence of said DNAse hypersensitivity site is indicative of the presence of beta thalassemia.
• the present invention relates to a composition for detecting or diagnosing beta thalassemia minor in a subject comprising a nucleic acid affinity ligand for one or more polymorphic sites as defined herein above.
• Fig. 2 provides an overall scheme of the genome wide association study for beta thalassemia minor.
• Fig. 6 shows haplotype blocks capturing associated SNPs.
• Fig. 6H shows the haplotype blocks of chromosome 12.
• indicator sequence refers to the sequence of an allele, which shows an association with a phenotype according to the present invention. Preferably, it shows an association with the phenotype of beta thalassemia.
• an indicator sequence may be not only the above indicated allelic sequence for each of SEQ ID NO: 1 to 14, but also an independent, further variation from the wildtype sequence as defined herein.
• SEQ ID NO: 3 defines a sequence of single nucleotide polymorphism (SNP) rs707497, which is located on chromosome 2, cytoband ql4.3, position 125064809 - 125065809 according to NCBI build 37.1 of the human genome, wherein at position 501 the wildtype nucleotide T is replaced by an indicator nucleotide, preferably by the nucleotide C.
• the SNP shows a minor allele frequency of 0.223300971.
• the SNP locus is located in the vicinity of gene CNTNAP5 at a distance of 282445 and 607555.
• SEQ ID NO: 10 defines a sequence of single nucleotide polymorphism (SNP) rsl6913719, which is located on chromosome 9, cytoband p21.1, position 28819174 - 28820174 according to NCBI build 37.1 of the human genome, wherein at position 501 the wildtype nucleotide C is replaced by an indicator nucleotide, preferably by the nucleotide T.
• the SNP shows a minor allele frequency of 0.14159292.
• the SNP locus is located in the vicinity of genes LOC646700 and MIRN876, at distances of -670440 and - 43949, respectively.
• SEQ ID NO: 12 defines a sequence of single nucleotide polymorphism (SNP) rsl7168572, which is located on chromosome 7, cytoband q21.3, position 97065519 - 97066519 according to NCBI build 37.1 of the human genome, wherein at position 501 the wildtype nucleotide A is replaced by an indicator nucleotide, preferably by the nucleotide G.
• the SNP shows a minor allele frequency of 0.133027523.
• the SNP locus is located in the vicinity of genes LOC442712 and TAC1, at distances of -235259 and - 295356, respectively.
• chromosome 1 for chromosome 1 : rsl 1573269 (SEQ ID NO: 9), rs4654885, rs441380, rsl0493137, rs6657279, rs6683003, rsl2082126, rsl529594, rsl2087676, rsl 1209819, rs576056, rsl 1808445, rs698944, rs291565, rsl7120268, rs41343145, rsl7018484, rsl6857061, rsl2131192, rsl2063296, rsl0913087, rs3009323, rs805911, rs6701222, rsl389970, rs6693224, rs6667309;
• the present invention relates to one or more, e.g. a panel, of the above mentioned polymorphic, changed sequences comprising the above mentioned indicator nucleotides, as constituting a marker for beta thalassemia.
• the term "marker for beta thalassemia” as used herein refers to the association of the mentioned SNP comprising the above identified indicator nucleotide at a sequence position as defined herein above in at least one allele, or, in specific embodiment, in two alleles of single subject, and the disease beta thalassemia.
• SNP rsl7024172 and SEQ ID NO: 5 with SNP rsl6950705 and SEQ ID NO: 6 with SNP rsl 1956461 and SEQ ID NO: 7 with SNP rs609539; or SEQ ID NO: 8 with SNP rs7975838 and SEQ ID NO: 9 with SNP rsl2063296 and SEQ ID NO: 10 with SNP rsl6913719 and SEQ ID NO: 11 with SNP rsl 1497898 and SEQ ID NO: 12 with SNP rsl7168572 and SEQ ID NO: 13 with SNP rsl6933412 and SEQ ID NO: 14 with SNP rsl6864505 etc. Further envisaged are all other 7 SNP permutations or groupings of the mentioned SNPs.
• SNP rsl7024172 and SEQ ID NO: 5 with SNP rsl6950705 and SEQ ID NO: 6 with SNP rsl 1956461 and SEQ ID NO: 7 with SNP rs609539 and SEQ ID NO: 8 with SNP rs7975838 and SEQ ID NO: 9 with SNP rsl2063296; or SEQ ID NO: 10 with SNP rsl6913719 and SEQ ID NO: 11 with SNP rsl 1497898 and SEQ ID NO: 12 with SNP rsl7168572 and SEQ ID NO: 13 with SNP rsl6933412 and SEQ ID NO: 14 with SNP rsl6864505 and SEQ ID NO: 1 with SNP rs666247 and SEQ ID NO: 2 with SNP rsl 2707034 and SEQ ID NO: 3 with SNP rs707497 and SEQ ID NO: 4 with SNP rsl 7024172 etc. Further envisage
• any possible 13 of the SNPs of the present invention e.g. SEQ ID NO: 1 with SNP rs666247 and SEQ ID NO: 2 with SNP rsl 2707034 and SEQ ID NO: 3 with SNP rs707497 and SEQ ID NO: 4 with SNP
• determining the nucleotide sequence at a polymorphic site refers to any suitable method or technique of detecting the identity of the nucleotide at position 501 of any one of or any grouping or panel comprising SEQ ID NO: 1 to 14. This determination method may predominantly be a sequencing technique or a technique based on complementary nucleic acid binding.
• the above mentioned sample is be a mixture of tissues, organs, cells and/or fragments thereof, or a tissue or organ specific sample, such as a tissue biopsy from vaginal tissue, tongue, pancreas, liver, spleen, ovary, muscle, joint tissue, neural tissue, gastrointestinal tissue, tumor tissue, or a body fluid, blood, serum, saliva, or urine.
• a tissue or organ specific sample such as a tissue biopsy from vaginal tissue, tongue, pancreas, liver, spleen, ovary, muscle, joint tissue, neural tissue, gastrointestinal tissue, tumor tissue, or a body fluid, blood, serum, saliva, or urine.
• blood sample comprising DNA-containing cells, e.g. non-matured red blood cells, erythrocyte precursor cells, leukocytes etc.
• bone marrow cells erythropoietic cells etc.
• the sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a way that nucleic acids
• the mentioned determination of the nucleotide sequence may be carried out through allele-specific oligonucleotide (ASO)-dot blot analysis, primer extension assays, iPLEX SNP genotyping, Dynamic allele-specific hybridization (DASH) genotyping, the use of molecular beacons, tetra primer ARMS PCR, a flap endonuclease invader assay, an oligonucleotide ligase assay, PCR- single strand conformation polymorphism (SSCP) analysis, quantitative real-time PCR assay, SNP microarray based analysis, restriction enzyme fragment length polymorphism (RFLP) analysis, targeted resequencing analysis and/or whole genome sequencing analysis.
• ASO allele-specific oligonucleotide
• primer extension assays iPLEX SNP genotyping
• DASH Dynamic allele-specific hybridization
• DASH Dynamic allele-specific hybridization
• molecular beacons tetra
• allele-specific oligonucleotide (ASO)-dot blot analysis refers to the employment of a short piece of synthetic DNA, which is typically complementary to the sequence of a polymorphic target site, in dot blot assay or,
• DASH genotyping refers to a technique taking advantage of the differences in the melting temperature in DNA that results from the instability of mismatched base pairs.
• a genomic segment may be amplified and attached to a bead through a PCR reaction, e.g. with a biotinylated primer.
• the amplified product may be attached to a streptavidin column and washed, e.g. preferably with NaOH, to remove the unbiotinylated strand.
• an allele-specific oligonucleotide may be added in the presence of a molecule that fluoresces when bound to double-stranded DNA. The intensity may
• the method as mentioned herein above may be combined with molecular functional analysis steps.
• the corresponding molecular pattern may additionally be analyzed in order to improve the diagnostic value of the method.
• the term "molecular pattern" as used herein refers to any suitable molecular or functional state, e.g. functional genomic state, which is linked to one or more of the SNPs of the present invention.
• SNPs of the present invention e.g. SNPs associated with SEQ ID NO: 1, 3, 5, 6, 7, 10, 11, 12, or 14 as defined herein above, which show no obvious functional relationship to a gene or regulatory region in the vicinity of said SNP may have a functional relation with respect to noncoding RNAs (Nardella C. et al, Curr Top Microbiol Immunol. 2010;347:135-68).
• noncoding RNAs may accordingly be detected with the help of suitable methods known to the person skilled in the art.
• such noncoding RNAs as well as repressor or activator factors thereof may be used for an improved diagnostic approach for the detection of beta thalassemia, or for a corresponding therapeutic approach.
• the nucleic acid affinity ligand may also be able to specifically bind to a DNA sequence being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 99.5% or 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequence of SEQ ID NO: 1 to 14, or fragments thereof, which comprise the polymorphic site as defined herein above, wherein said sequence of SEQ ID NO: 1 to 14 comprises the respective indicator nucleotide as described herein above, or to any fragments of said sequences.
• said nucleic acid affinity ligand may be a short nucleic acid molecule, e.g. a RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule or any other suitable nucleic acid format known to the person skilled in the art, being capable of specifically binding to the sequence of SNPs (e.g. indicator or wildtype sequence) of SEQ ID NO: 1 to 14.
• the present invention relates to the use of a nucleic acid molecule as defined herein above for detecting or diagnosing beta thalassemia in a subject.
• the present invention relates to the use of a nucleic acid molecule as defined herein above for detecting or diagnosing beta thalassemia minor in a subject.
• a nucleic acid molecule as defined herein above may be used as a template for a corresponding detection approach, e.g. based on the above defined methods. More preferably an affinity ligand for a polymorphic site according to the present invention, e.g.
• a oligonucleotide or probe may be employed in a suitable method for the detection of the presence of a wildtype or indicator nucleotide at position 501 of SEQ ID NO: 1 to 14.
• the presence of beta thalassemia preferably of beta thalassemia minor, may be confirmed or denied as described herein above.
• the kit may also comprise accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes, or other suitable compound or liquids necessary for the performance of a nucleic acid detection.
• accessory ingredients like secondary affinity ligands, e.g. secondary antibodies, detection dyes, or other suitable compound or liquids necessary for the performance of a nucleic acid detection.
• Such ingredients as well as further details would known to the person skilled in the art and may vary depending on the detection method carried out.
• the kit may comprise an instruction leaflet and/or may provide information as to the relevance of the obtained results.
• Samples were selected based on a number of parameters which includes: a. Ethnicity - Samples were collected from the North Indian population b. Sex
• Nsp RE digestion genomic samples are digested with Nsp I restriction enzyme using the same digestion protocol.
• Affymetrix GeneChip Command Console maps the pixel intensity to probe annotation (supplied by Affymetrix) to generate .CEL files that contain the signal values for the probe. 5.
• the .CEL files for each individual were subjected to quality control (QC). Some examples of various QCs performed on the samples are given in Table 2. Genotyping Console(GTC) was used to perform QC using the following metrics:
• carboxypeptidase A6 ///NM 001127445 // intron // 0 // Hs.658850 // CPA6 // 57094 // carboxypeptidase A6 /// ENST00000297769 // intron // 0 //Hs.658850 // CPA6 // 57094 // carboxypeptidase A6 /// ENST00000297770 // intron // 0 //Hs.658850 // CPA6 // 57094 // carboxypeptidase A6
• 154141 // membrane bound O-acyltransferase domain containing 1 /// NM_001546 //downstream // 192545 // Hs.519601 // ID4 // 3400 // inhibitor of DNA binding 4, dominant negative helix-loop-helix protein /// ENST00000324607 //downstream // 67475 //Hs.377830 // MBOAT1 // 154141 // membrane bound O-acyltransferase domain containing 1 /// ENST00000378700 //downstream // 192545 // Hs.519601 // ID4 // 3400 // inhibitor of DNA binding 4, dominant negative helix-loop-helix protein rs707497 NMJ30773 // intron // 0 // Hs.660653 // CNTNAP5 // 129684 // contactin associated protein-like 5 /// ENST00000285362 // intron // 0 //Hs.660653 // CNTNAP5 // 129684 // contactin associated protein-like 5 rs7975838 NR_027345 // up

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