Are backup BM harvests worthwhile in unrelated donor allogeneic transplants?

The Cleveland Clinic blood and marrow transplant program has routinely performed 'backup' autologous harvests in unrelated recipients with hematological malignancies in remission, lymphoma without marrow involvement and CML in chronic phase. We reviewed all adult or cord unrelated donor (URD) transplants performed from January 1995 through September 2008 to evaluate the value of this procedure. Of 130 patients who had backup harvests, 15 (11%) had their backup harvests re-infused, all for graft failure. No patients undergoing fully ablative preparation and unmanipulated or T-depleted grafts from well-matched adult donors required infusion of backup marrow. Nine of 42 patients who underwent T cell grafts from partially matched or mismatched donors, five patients undergoing partially matched ablative transplants from adult donors or cord blood, and one patient undergoing non-myeloablative transplant required infusion of their back-up harvest. Five of 15 patients who received their backup marrow are alive in CR 2-11.6 (median 7.6) years from infusion. Two of these five were bridged to a second URD transplant; the other three showed durable disease-free survival without a second allogeneic transplant. Backup harvest is unnecessary for HLA well-matched myeloablative transplants, but may be useful in patients at higher risk of graft failure.

Restricted tissue expression pattern of a novel human rasGAP-related gene and its murine ortholog.

The mammalian rasGAPs constitute a group of widely expressed proteins involved in the negative regulation of ras-mediated signaling. In this study we have isolated a novel human gene, RASAL (Ras GTPase-activating-like) and its murine ortholog, MRASAL which are most similar to the GAP1 family of rasGAP proteins, based upon the presence and organization of specific conserved domains. Full-length human and murine mRNA sequences are predicted to encode 804 and 799 amino acid polypeptides, respectively. Sequence analysis of these two proteins revealed the presence of two N-terminal calcium-dependent phospholipid binding C2 domains, a conserved GAP related domain (GRD) and a C-terminal pleckstrin homology (PH) domain. Northern blot and mRNA in situ hybridization analyses indicate that RASAL, in contrast to other mammalian rasGAP proteins, has a limited expression pattern; RASAL is highly expressed in the follicular cells of the thyroid and the adrenal medulla and expressed at lower levels in brain, spinal cord and trachea. Human RASAL has been localized by radiation hybrid mapping to chromosome 12q23-24.

An expression-independent catalog of genes from human chromosome 22.

To accomplish large-scale identification of genes from a single human chromosome, exon amplification was applied to large pools of clones from a flow-sorted human chromosome 22 cosmid library. Sequence analysis of more than one-third of the 6400 cloned products identified 35% of the known genes previously localized to this chromosome, as well as several unmapped genes and randomly sequenced cDNAs. Among the more interesting sequence similarities are those that represent novel human genes that are related to others with known or putative functions, such as one exon from a gene that may represent the human homolog of Drosophila Polycomb. It is anticipated that sequences from at least half of the genes residing on chromosome 22 are contained within this exon library. This approach is expected to facilitate fine-structure physical and transcription mapping of human chromosomes, and accelerate the process of disease gene identification.

A 500-kilobase region containing the tuberous sclerosis locus (TSC1) in a 1.7-megabase YAC and cosmid contig.

A complete overlapping clone map of a 1.7-Mb region from DBH to D9S67 that includes the TSC1 candidate region has been constructed. The map includes YAC and cosmid clones, contains STS approximately every 50 kb on average, and establishes the order of five previously unordered loci. The overall physical length of this segment of chromosome 9q34 (1.7 Mb) is significantly less than expected compared to its estimated genetic length (approximately 10 cM). Consequently, the physical length of the TSC1 candidate region is substantially less than predicted by a genetic distance of approximately 2 cM.

Isolation of genes from complex sources of mammalian genomic DNA using exon amplification.

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.