Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A.

A syndrome of lymphoblastic lymphoma, eosinophilia, and myeloid hyperplasia/malignancy associated with t(8;13)(p11;q11): Description of a distinctive climicopathologic entity. Simultaneous occurrence of a T-cell lymphoma and chronic myeloid leukemia with an unusual karyotype. translocation breakpoints associated with an atypical myeloproliferative disorder: Evidence for three discreet loci involved in myeloid leukemias on 8p11. associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11-12.: FGFR1 is fused with a novel zinc-finger gene, ZNF198, in the t(8;13) leukemia/lymphoma syn-J: The t(8;13)(p11;q11-12) rearrangement associated with an atypical myeloproliferative disorder fuses the fibroblast growth factor receptor 1 gene to a novel gene RAMP. factor receptor 1 is fused to FIM in stem-cell myeloproliferative disorder with t A YAC contig and an EST map in the pericentromeric region of chromosome 13 surrounding the loci for neurosensory nonsyndromic deafness (DFNB1 and DFNA3) and limb-girdle muscular dystrophy type 2C (LGMD2C).(p11;q12) translocation from a patient with myeloproliferative disease using fluorescence in situ hybridization. Hemophilia A is the most common X-linked coagulation disorder, with an incidence of about one in 5,000 males. About half the families with severe disease have a large genomic inversion of the factor VIII gene that separates the first 22 exons from the final 4 exons. This inversion results from a hotspot of recombination between a 9.5-kb region in intron 22 (int22h1) and either of two extragenic, distal homologs, int22h2 and int22h3; int22h2 and int22h3 are more than 99% identical to one another. Recombination produces an inversion because the extragenic homologs are in the opposite orientation relative to int22h1. 1,2 The inversions are detected by Southern blotting, which is slow and labor-intensive. A rapid and inexpensive test is of particular clinical utility, because carrier testing is often paid out-of-pocket due to insurance issues and confidentiality; a low-cost test may facilitate more optimal use of genetic services. This difficult 9.5-kb region previously has been refractory to polymerase chain reaction (PCR) amplification, presumably due to the presence of a 3.5-kb GC island, which includes a 1-kb segment with a GC content of 79%. In addition, an optimal PCR-based assay requires more genomic sequence flanking the ho-mologs. We have developed a single-tube PCR assay that combines overlapping PCR 3 with long-distance PCR 4 to achieve the genetic diagnosis of inversions causing hemophilia A (Fig 1). The inversion was detected by performing PCR directly from genomic DNA with four primers that differentiate the wild-type, inversion, and …