Reprint requests to: Ian J. Cohen, M.B., Ch.B.,The National Center for Pediatric, Hematology/Oncology, Schneider Children's Medical Center of Israel, Beilinson Campus, Petah Tiqva, 49202 ISRAEL, phone: 972-3-9253781, fax: 972-3-9253042, e-mail: icohen@ccsg.tau.ac.il.

ABSTRACT. Partial splenectomy for Gaucher disease is often followed by reenlargement of the splenic remnant. It remains unclear if this process is due to tissue regeneration or to continued deposition of glucocerebroside in the reticuloendothelial system or both. We compared the splenic architecture before and after reenlargement in three cases of failed repeated partial splenectomy after two, six and five years. Using the number of lymphoid follicles per hundred low power fields (LF/LPF) as an arbitrary index, we found that prior to the first operation 18, 20 and 27 lymphoid follicles were present per one hundred low power fields, while at the second operation, the corresponding rates were 11, 15 and 17; in control spleens, an average of 712.5 lymphoid follicles were present in one hundred low power fields. The difference in the LF/LPF ratio before and after reenlargement, led us to speculate that splenic re-enlargement in Gaucher disease is mainly the result of the continued deposition of the glucocerebroside in the reticuloendothelial system of the splenic remnant, though some degree of true regeneration as well cannot be completely ruled out. These findings are compared with animal studies and results for partial splenectomy on humans, performed for trauma. Further studies in patients with Gaucher disease are warranted to better define the underlying mechanism of splenic reenlargement.

Keywords: Gaucher disease, splenic regeneration, partial splenectomy.

Reprint requests to: Enrique Freud, M.D., Department of Pediatric Surgery, Schneider Children's Medical Center of Israel, Beilinson Campus, Petah Tiqva 49202, Israel, phone 972-3-925 3741, fax 972-3-9253899, e-mail: nsfre@netvision.net.il.

ABSTRACT. The Cys282->Tyr mutation in the HFE gene is carried by the majority of hereditary hemochromatosis patient chromosomes, yet some patients do not seem to harbor any mutation in this gene. This suggests a possibility that these patients may have a mutation in other genes in the same pathway as HFE. We analyzed the cDNA sequences of transferrin receptor (TFR), which was recently shown to interact with HFE, in twenty-one hereditary hemochromatosis patients including sixteen individuals who did not carry a Cys282->Tyr mutation. A nucleotide substitution (424A->G), which resulted in the Ser142->Gly amino acid substitution, was the only amino acid polymorphism detected in the open reading frame of the TFR gene in these patients. This amino acid substitution was a rather common polymorphism in the general population (49%) and its frequency did not significantly differ in the hereditary hemochromatosis (HH) patients regardless of the HFE genotype. Thus, amino acid changes in the TFR gene do not appear to play a role in HH even when the patients do not have a HFE mutation. However, this study does not rule out the possibility of the involvement of mutations in non-coding regions.

Keywords: hereditary hemochromatosis, HFE, transferrin receptor, polymorphism

Reprint requests to: Zenta Tsuchihashi, Ph.D., Progenitor, Inc., 4040 Campbell Ave., Menlo Park, CA 94025, USA, phone (650)614- 7035, fax (650)617-0883 e-mail: zenta@progenitor.com.

ABSTRACT. Retrovirus vectors for Agamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha- globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various Agamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, Agamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the betapr.Agamma cassettes.

Keywords: gene regulation, globin, locus control region, expression, gene therapy, retrovirus, hematopoietic, cell line

Reprint requests to: George Stamatoyannopoulos, M.D., Dr.Sci., Professor of Medicine, Head, Division of Medical Genetics, University of Washington, Box 357720, Seattle, WA 98195, phone (206)543-3526, fax (206)543-3050 e-mail: gstam@u.washington.edu.