Mutant calreticulin for the diagnosis of myeloid malignancies

Abstract

The present invention relates to a method for diagnosing myeloid malignancy comprising determining the presence of a mutant allele of the calreticulin gene. Also genomic sequences, cDNA sequences, mRNA sequences and protein sequences of the mutant calreticulin are subject of the present invention. Further, the invention relates to medical uses of inhibitors of mutant calreticulin.

1 - 88 . (canceled) 
     
     
         89 . A composition comprising at least one labelled polynucleotide, wherein said polynucleotide detects the presence of one or more mutations in the calreticulin exon 9 nucleic acid sequence of SEQ ID NO:435 in a sample isolated from bone marrow, blood or saliva. 
     
     
         90 . The composition of  claim 89 , wherein the mutated calreticulin allele is detected by sequencing the exon 9 nucleic acid sequence. 
     
     
         91 . The composition of  claim 89 , wherein the mutated calreticulin allele is detected by fragment analysis of the exon 9 nucleic acid sequence. 
     
     
         92 . The composition of  claim 89 , wherein the mutated calreticulin allele is detected by both sequencing the exon 9 nucleic acid sequence and by fragment analysis of the exon 9 nucleic acid sequence. 
     
     
         93 . The composition of  claim 90 , wherein sequencing is performed by Sanger sequencing or bidirectional Sanger sequencing. 
     
     
         94 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence comprises insertions in SEQ ID NO:435. 
     
     
         95 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence comprises deletions in SEQ ID NO:435. 
     
     
         96 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence comprises a frameshift mutation in SEQ ID NO:435. 
     
     
         97 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence is amplified with primers that specifically hybridize to exon 9 of the calreticulin gene and determining the size of the amplified nucleic acid sequence. 
     
     
         98 . The composition of  claim 97 , wherein amplifying is performed by polymerase chain reaction (PCR). 
     
     
         99 . The composition of  claim 97 , wherein determining the size of the amplified nucleic acid sequence is performed by a sizing assay. 
     
     
         100 . The composition of  claim 99 , wherein the sizing assay is electrophoresis. 
     
     
         101 . The composition of  claim 89 , wherein the one or more mutations in the exon 9 nucleic acid sequence is detected by any one or more of: random amplified polymorphic detection (RAPD), amplified fragment length polymorphism detection (AFLPD), allele specific oligonucleotide (ASO) probes, TaqMan probe principle, hybridization to DNA microarrays or beads, and/or high resolution melting (HRM). 
     
     
         102 . The composition of  claim 89 , wherein the sample is a bone marrow sample or a blood sample. 
     
     
         103 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence is derived from genomic DNA. 
     
     
         104 . The composition of  claim 89 , wherein the exon 9 nucleic acid sequence is derived from mRNA. 
     
     
         105 . The composition of  claim 104 , wherein the presence of the mRNA is determined by RealTime PCR, ReverseTranscriptase PCR, Whole Transcriptome Shotgun Sequencing (RNAseq), in situ hybridization or micro-arrays. 
     
     
         106 . The composition of  claim 89 , wherein the nucleic acid sequence is derived from cDNA. 
     
     
         107 . The composition of  claim 89 , wherein the mutated calreticulin allele is detected by contacting a nucleic acid molecule from the sample with the labelled polynucleotide that specifically hybridizes to a mutated sequence in exon 9 of the calreticulin gene of SEQ ID NO:435. 
     
     
         108 . The composition of  claim 107 , wherein the nucleic acid molecule is produced from an amplification reaction. 
     
     
         109 . The composition of  claim 108 , wherein the labelled polynucleotide is labelled using a fluorescent label or dye. 
     
     
         110 . The composition of  claim 89 , wherein the nucleic acid molecule comprises the nucleic acid sequence as shown in SEQ ID NO: 145, 146, 147, 149, 150, 151, 153, 154, 155, 157, 158, 159, 161, 162, 163, 165, 166, 167, 169, 170, 171, 173, 174, 175, 177, 178, 179, 181, 182, 183, 185, 186, 187, 189, 190, 191, 193, 194, 195, 197, 198, 199, 201, 202, 203, 205, 206, 207, 209, 210, 211, 213, 214, 215, 217, 218, 219, 221, 222, 223, 225, 226, 227, 229, 230, 231, 233, 234, 235, 237, 238, 239, 241, 242, 243, 245, 246, 247, 249, 250, 251, 253, 254, 255, 257, 258, 259, 261, 262, 263, 265, 266, 267, 269, 270, 271, 273, 274, 275, 277, 278, 279, 281, 282, 283, 285, 286, 287, 291, 292, 293, 295, 296, 297, 299, 300, 301, 303, 304, 305, 307, 308, 309, 311, 312, 313, 315, 316, 317, 319, 320, 321, 323, 324, 325, 327, 328, 329, 331, 332, 333, 335, 336, 337, 339, 340, 341, 343, 344, 345, 347, 348, 349, 351, 352, 353, 355, 356, 357, 359, 360, 361, 363, 364, 365, 367, 368, 369, 371, 372, 373, 375, 376, 377, 379, 380, 381, 383, 384, 385, 387, 388, 389, 391, 392, 393, 395, 396, 397, 399, 400, 401, 403, 404, 405, 407, 408, 409, 411, 412, 413, 415, 416, 417, 419, 420, 421, 423, 424, 425, 427, 428, 429, 431, 432, or 433.