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We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from 1968 until 1992. The patients received a graft from an HLA-identical related (n = 10), an HLA–haplo-identical related (n = 19), or a closely HLA-matched unrelated (n = 2) donor that resulted in the long-term survival of 6 of 10, 9 of 19, and 0 of 2 children, respectively. Major complications included failure of engraftment and early death caused by respiratory failure. The chimerism pattern and immunologic reconstitution were evaluated in 15 children who survived more than 1 year with sustained engraftment. The pattern of engraftment was investigated within flow-sorted peripheral blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain reaction. The immunologic reconstitution was assessed by various in vitro and in vivo parameters. Although the number of PB T cells and the in vitro T-cell proliferative response was in the lower region of normal in the majority of cases and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% to 5%) were detected in eight cases at multiple occasions. All children showed normal serum IgM and IgG subclass levels and produced specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical graft recipients with host-type B lymphoid and myeloid cells have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin. Donor NK cell engraftment restored defective in vitro NK cell function of the recipient. We conclude that determination of lineage-specific engraftment patterns provides valuable information for the understanding of the immunologic reconstitution after allogeneic BMT for SCID.

A genetic linkage study was performed in a large Dutch kindred with erythrokeratodermia variabilis (EKV, McKusick no. 13320). The autosomal-dominant trait appeared to segregate rather consistently with the cde (r) gene complex of the Rh system. Only one recombinant was found amongst 27 informative individuals. Lod score calculations gave strong evidence for close linkage between the loci for EKV and Rh (with a maximum lod score of 5.55 at a recombination fraction of 0.044).

Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogenesis. For the identification of protein truncating mutations of the APC gene, we developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectiveness of this method. Copyright 1994, 1999 Academic Press

Techniques are described for the electrophoretic characterization on Cellogel of adenosine deaminase, adenylate kinase, enolase, fumarase, a-galactosidase, glucosephosphate isomerase, glutathione reductase, glutamic-oxaloacetic transaminase, guanylate kinase, hexosaminidase, hypoxanthine-guanine phosphoribosyltransferase, indophenol oxidase, “malic” enzyme, mannosephosphate isomerase, nucleoside phosphorylase, peptidase-A, peptidase-B, peptidase-C, phosphoglucomutase, pyruvate kinase, triosephosphate isomerase, and UDP glucose pyrophosphorylase1 in the man-Chinese hamster somatic cell hybrid systems.

A simple, fast and direct staining method for the detection of hypoxanthineguanine phosphoribosyltransferase is described. It is based on the conversion of inosine monophosphate to hypoxanthine, which is then enzymatically oxidized. This oxidation is coupled to the reduction of a tetrazolium salt to blue formazan. The electrophoretic pattern of HGPRT in man-Chinese hamster hybrid cells shows a three-banded pattern, suggesting a dimeric structure for this enzyme.

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). Genotype-phenotype correlation studies in patients with FAP have demonstrated associations of certain variants of the disease with mutations at specific sites within the APC gene. In a large FAP family, we identified a frameshift mutation located in the alternatively spliced region of exon 9. Phenotypic studies of affected family members showed that the clinical course of FAP was delayed, with gastrointestinal symptoms and death from colorectal carcinoma occurring on average 25 and 20 years later than usual, respectively. The numbers of colorectal adenomas differed markedly among affected individuals and the location of colorectal cancer lay frequently in the proximal colon. Our findings suggest that the exon 9 mutation identified in the pedigree is associated with late onset of FAP. The atypical phenotype may be explained by the site of the mutation in the APC gene. Analysis of the APC protein product indicated that the exon 9 mutation did not result in a detectable truncated APC protein. Given the location of the mutation within an alternatively spliced exon of APC, it is conceivable that normal APC proteins are produced from the mutant allele by alternative splicing.

A specific competitive radioimmunoassay (RIA) was employed to quantify human adenosine deaminase molecules produced in human-Chinese hamster somatic cell hybrids. Studies on a set of hybrids in which the normal and aberrant expressions of adenosine deaminase (assigned earlier to human chromosome 20) were segregating, have demonstrated that in the patient with ADA -SCID disease reported by Herbschleb-Voogt et al (1981a), the deficiency of ADA activity was associated with a comparable deficiency of adenosine deaminase specific immuno-crossreacting material (ADA-CRM).

The localization of the structural gene for human a-galactosidase B (=N-acetyl-a-galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman a-galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl-a-galactosominidase. The results show that the structural gene for human a-galactosidase B is situated on chromosome 22, and that there is no structural relationship between human a-galactosidase A and human a-galactosidase B.

An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.

In man-Chinese hamster somatic cell hybrids the segregation of the loci for 27 human enzyme markers and the species-specific surface antigens, including the HL-A histocompatibility antigens, was studied. The results show a synteny of the human loci for phosphoglucomutase 3, cytoplasmic malic enzyme, tetrameric indophenol oxidase, and HL-A. Furthermore, evidence is presented that the loci for the human species-specific antigens are distributed over several chromosomes.

THE existence of zymographically detectable interspecific enzyme variations and the preferential loss of human chromosomes in the man–mouse as well as the man–Chinese hamster somatic cell hybrids have rendered these hybrids useful for genetic analysis of man1,2. Of these two systems, linkage studies involving the human locus for 6-phosphogluconate dehydrogenase, 6PGD, have been found feasible only in the man–Chinese hamster hybrids because the usual phenotype of human 6PGD is electrophoretically distinguishable only in this hybrid system3. This report deals with the genetic linkage relations of the human 6PGD locus in man–Chinese hamster somatic cell hybrids and provides the first evidence that the human genes for 6PGD and phosphoglucomutase1(PGM1) are syntenic (located on the same chromosome)4.