Medizin - Open Access LMU - Teil 08/22 Archiv

Two patients with a history of penile prosthesis removal presented for non-prosthetic treatment of their erectile dysfunction. The first patient had a penile implant for two years before it was removed and showed extensive cavernosal fibrosis. He did not respond to intracavernosal injection of vasoactive drugs. The second patient had the prosthesis for four weeks. He showed no evidence of cavernosal fibrosis and responded well to the intracavernous injection. He has been in an iniracavernous autoinjection program for more than one year without complications.

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.

Chromosomal in situ suppression (CISS)-hybridization of biotinylated phage DNA-library inserts from sorted human chromosomes was used to decorate chromosomes 1 and 7 specifically from pter to qter and to detect structural aberrations of these chromosomes in irradiated human peripheral lymphocytes. In addition, probe pUC1.77 was used to mark the Iq12 subregion in normal and aberrant chromosomes 1. Low LET radiation (60Co--rays; 1.17 and 1.33 MeV) of lymphocyte cultures was performed with various doses (D = 0, 2, 4, 8 Gy) 5 h after stimulation with phytohaemagglutinin. Irradiated cells were cultivated for an additional 67 h before Colcemid arrested metaphase spreads were obtained. Aberrations of the specifically stained chromosomes, such as deletions, dicentrics, and rings, were readily scored after in situ hybridization with either the 1q12 specific probe or DNA-library inserts. By the latter approach, translocations of the specifically stained chromosomes could also be reliably assessed. A linear increase of the percentage of specifically stained aberrant chromosomes was observed when plotted as a function of the square of the dose D. A particular advantage of this new approach is provided by the possibility to delineate numerical and structural chromosome aberrations directly in interphase nuclei. These results indicate that cytogenetic monitoring of ionizing radiation may be considerably facilitated by CISS-hybridization.

Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence — polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.