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Background: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector.Methods: Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery.Findings: All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes.Interpretation: Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity. [ABSTRACT FROM AUTHOR]

We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Mutations in the Bruton's tyrosine kinase (BTK) gene result in XLA. Despite the large numbers of BTK mutations reported, no correlation can be made between the clinical phenotype and the gene defects. Analysis of Btk protein expression and activity in individuals with XLA was performed to characterize the relationship between a particular mutation, the resultant Btk protein and the clinical phenotype. In most patients studied, including those with atypical phenotypes, there was complete absence of protein expression and activity. Furthermore, in two undiagnosed individuals with a clinical phenotype suggestive of XLA, lack of protein expression was used to confirm an abnormality in Btk. These results underline the importance of protein analysis prior to speculating on protein structure and function based on the gene mutation. Lack of Btk expression in atypical phenotypes suggests that there is redundancy in B lymphocyte signalling such that alternative signalling molecules, or mechanisms, can compensate for the lack of Btk. We also suggest that analysis of Btk expression can be used as an indicator of XLA. These rapid assays may be used to screen a wider spectrum of individuals with humoral immunodeficiency in order to characterize fully the extent of Btk deficiency.

Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.

Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling.

Gene transfer to photoreceptor cells may provide a means for arresting the retinal degeneration that is characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). However, transduction of photoreceptors has to date been inefficient, and further limited by toxicity and immune responses directed against vector-specific proteins. An alternative vector system based on adeno-associated virus (AAV) may obviate these problems, and may be useful for transduction of neuronal cells. In this study we have demonstrated successful transduction of all layers of the neuroretina as well as the retinal pigment epithelium (RPE) following subretinal injection of recombinant AAV particles encoding lac Z. Furthermore, the efficiency of transduction of photoreceptors is significantly higher than that achieved with an equivalent adenoviral vector. This is the first report showing that AAV is capable of transducing photoreceptor cells and supports the use of this vector system for gene therapy of retinal diseases such as RP.

Adenosine deaminase (ADA) deficiency results in severe combined immune deficiency disease (SCID), which is fatal without treatment. Allogeneic bone marrow transplantation (BMT) is the treatment of choice if an HLA-identical sibling bone marrow donor is available, resulting in almost 100% cure rate. BMT-related mortality is high in patients lacking such a donor. For these patients, efficient transfer of a recombinant ADA gene into hematopoietic stem cells is a therapeutic option if it results in the outgrowth of a 'genetically repaired' lymphoid system. Based on successful gene transfer studies in monkeys, we performed retrovirus-mediated gene transfer into CD34+ bone marrow cells of three patients with ADA deficiency. Two patients received bovine ADA conjugated to polyethylene glycol (PEG-ADA); in the third patient, PEG-ADA was started 4 months after gene transfer. Gene transfer resulted in a 5-12% transduction frequency of in vitro colony forming cells (CFU-Cs). No toxicity was observed during and after infusion of the graft. Following infusion of the transduced CD34+ cells, transduced granulocytes and mononuclear cells persisted in the circulation for 3 months. In addition, the gene was present in the marrow of one of the patients at 6 months after gene transfer. Expression of the gene was not detected. After this period, the gene could not be detected. In monkey studies we showed that myeloablation, which was not performed in the patients, may enhance engraftment of genetically modified cells. We hypothesize that lack of myeloablation, administration of bovine ADA and low numbers of transduced progenitor cells all may have contributed to the relative low numbers of transduced cells in the patients. Under these conditions, no selective advantage of the genetically corrected progenitor cells was observed.

Mutations in the common gamma chain (gamma c or IL2RG) of the interleukin-2, -4, -7, -9 and -15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the gamma c proteins produced as a result of these mutations are discussed.

The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).

X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain.

For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential.

X-linked agammaglobulinemia is a primary inherited immunodeficiency resulting in a lack of or dramatic reduction in the number of mature B lymphocytes and, thus, greatly reduced levels of serum immunoglobulin. The defect results from mutations in the gene for Bruton's tyrosine kinase (Btk). Using rabbit antisera generated against Btk, we have demonstrated an increase in the level of in vitro kinase activity present in anti-Btk immunoprecipitates from B cells following stimulation with anti-immunoglobulin antibody. This increase in immune complex kinase activity is detectable 1 to 2 min following stimulation and remains elevated for over 30 min. A similar increase was not seen with two late pre-B cell lines investigated in the same way. This stimulation of activity may suggest a role for Btk in signalling through the B cell receptor or associated proteins, in mature B cells.

Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X-linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre-B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein-Barr virus-transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil-derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.

We have produced physical maps of the proximal part of Xq22, containing the Bruton's tyrosine kinase (BTK) and alpha-galactosidase A (GLA) gene loci, using long range physical mapping techniques and yeast artificial chromosomes (YACs). These maps reveal five previously unidentified CpG islands which could indicate the presence of other genes in this region.

We describe a family affected by X linked severe combined immunodeficiency (SCIDX1) in which genetic prediction of carrier status was made using X chromosome inactivation studies together with limited genetic linkage analysis. Linkage studies in this family showed a confusing pattern of inheritance for the X chromosome. A female with a random pattern of X chromosome inactivation in her T cells appeared to have inherited an X chromosome with four recombinations within 10 cM. The odds of this happening in a single meiotic event make this an unlikely explanation. Data obtained from studying the X chromosomes of her two unaffected sons showed that this could be explained simply on the basis of her having inherited three alleles each of the relevant polymorphic DNA loci. We used fluorescent in situ hybridisation (FISH) to confirm that this person had inherited three complete X chromosomes. Thus, although the results from X chromosome inactivation analysis indicated that this subject was not a carrier of the affected chromosome, FISH and genetic linkage analysis showed clearly that the affected chromosome had been inherited. The implications of this finding for diagnosis of carrier status in this family and for other families with X linked inherited immunodeficiencies is discussed.

The gene which causes X-linked agammaglobulinemia, btk, has recently been identified as a cytoplasmic tyrosine kinase expressed almost exclusively in B cells, and at all stages of B-cell differentiation. To assess the possibility of involvement of this gene in childhood B-cell malignancies, cells from 23 pediatric patients with B-cell acute lymphoblastic leukemia were examined for expression and alteration of the Btk protein and also for mutations in the btk gene. Btk proteins, similar in both molecular weight and quantity to those seen in unaffected individuals, were detected in whole cell lysates from the blasts of 12/12 patients indicating that no abnormal protein was present. cDNAs from the leukemic blasts of all 23 patients were screened with specific primers covering the coding region of the btk cDNA for mutations using single strand conformation polymorphism (SSCP) analysis. No mutations were found but a nucleotide polymorphism was identified in 4/23 patients at the 3' end of btk. Although the sample size in this study was relatively small, these data suggest that btk does not appear to play a critical role in childhood B-cell leukemias.

We investigated whether rhesus monkey CD34+CD11b- hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b- cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marrow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b- bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency.

The gene defective in X-linked agammaglobulinemia (XLA) has recently been isolated and identified as btk, a non-receptor protein tyrosine kinase. We have utilized the technique of single strand conformation polymorphism (SSCP) analysis for the btk gene to identify mutations in XLA patients. The btk gene in affected boys from 10 families was analysed and mutations were identified in eight cases; seven of these were point mutations and one was a small insertion. The mutations were found throughout the gene coding region. Six of the patients have classical XLA and two have less severe forms of the disease. We have also identified a polymorphism at nucleotide position 2031. This technique will allow us to provide more accurate diagnoses of the disease and to determine the nature of the functional defects in the btk gene in these families.

The dinucleotide repeat sequences at the DXS454 and DXS458 loci have been mapped genetically to Xq22, to the interval between DXS3 and DXS17. We have now mapped them with respect to XLA and five other loci, to within the DXS3 to XLA interval. The more precise localisation of these polymorphic loci will be useful for the fine-mapping of disease loci on the long arm of the X chromosome and enable these probes to be used for prenatal diagnosis and carrier status determination in families with XLA.

We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.

The btk gene has recently been identified as the causative gene in X-linked agammaglobulinemia (XLA). This has opened up many new possibilities for the treatment of this B-cell immunodeficiency. Christine Kinnon and colleagues review the high degree of sequence of homology of btk to the non-receptor tyrosine kinases and speculate on putative roles for this gene in B-cell development.

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.

A number of human disease genes have been localised to Xq12-21.1. A genetic map of this region has previously been constructed using family linkage studies and has been complemented by physical mapping studies using hybrid and deletion cell lines. We have constructed a preliminary long-range physical map of the region, which incorporates thirteen polymorphic and non-polymorphic probes, using pulsed field gel electrophoresis. The order of loci that can be inferred from all the genetic and physical mapping data is: cen-DXS133-[DXS153, DXS159]-DXS132-DXS135-[DXS131, DXS162]-[DXS325, DXS-347, DXS441]-PGK1-DXS447-DXS72-tel. The detection of several large non-overlapping MluI fragments by these probes implies that the minimum extent of the genomic DNA containing these loci is 16 Mb. This information should be useful in the eventual identification and isolation of the genes responsible for diseases that map to this region.

The gene responsible for X-linked agammaglobulinemia (XLA) has not been identified; however, in the course of genetic linkage studies designed to map the locus more precisely, a number of closely linked polymorphic loci have been identified. These have proved to be useful in identifying carriers and in pre-natal diagnosis of this disease. The DXS178 locus was found to be closest to the XLA locus and has been the most usefully employed probe to date. Using physical mapping techniques, we have identified a previously cloned genetic marker, DXS265, as being situated within 5 kb of DXS178. So far, we have found one family that is not informative for DXS178 but that is informative for DXS265; females in this family can now be offered the possibility of carrier determination and pre-natal diagnosis for this life-threatening disease.

X linked immunodeficiency with hyperimmunoglobulinaemia M (HIGM1), which is characterised by agammaglobulinaemia together with excess IgM production reflecting an impairment of the immunoglobulin heavy chain class switch of B lymphocytes, has been mapped to Xq26. We report multipoint linkage data in six families with HIGM1 which show that the most likely position for the gene is close to HPRT with a maximum lod score of 4.89. The finding of recombinations between HIGM1 and both HPRT and DXS42 implies that HIGM1 is not allelic to X linked lymphoproliferative disease. These data will be useful in genetic counselling in families and will also be useful in testing candidate genes.

X chromosome-linked immunodeficiency with hyper-IgM (HIGM1, MIM number 308230) is a rare disorder characterized by recurrent bacterial infections, very low or absent IgG, IgA and IgE, and normal to increased IgM and IgD serum levels. HIGM1 has been suggested to result from ineffective T-cell help for B cells. We and others have identified a novel, TNF-related activation protein (TRAP) that is exclusively expressed on the surface of stimulated T cells. TRAP, a type II transmembrane protein of M(r) 33,000, is the physiological ligand for CD40 (refs 5-8). Crosslinking of CD40 on B cells induces, in the presence of lymphokines, immunoglobulin class switching from IgM to IgG, IgA or IgE. Mapping of the TRAP gene to the X-chromosomal location q26.3-q27.1 (ref. 6) suggested a causal relationship to HIGM1, which had previously been assigned to Xq26 (refs 12-14). Here we present evidence that point mutations in the TRAP gene give rise to nonfunctional or defective expression of TRAP on the surface of T cells in patients with HIGM1. The resultant failure of TRAP to interact with CD40 on functionally intact B cells is responsible for the observed immunoglobulin isotype defect in HIGM1.

The Xq22 region of the human X chromosome encompasses the loci of several genes and random DNA markers whose relative positions have not been determined. By a combination of PFGE mapping and the analysis of a selected panel of X chromosome radiation hybrid cell lines, we have constructed physical maps of Xq22 that order a total of 11 polymorphic and nonpolymorphic DNA markers. Ten of these probes have been linked physically into three separate clusters, spanning nearly 6 Mb of DNA in total. The DXS94, DXS147, DXS211, DXS17, and DXS87 loci are all present on a 2.7-Mb MluI fragment; PLP, DXS54, DXS24, and DXS83 are present on MluI fragments spanning over 1.6 Mb; and DXS178 is present on a 1.5-Mb MluI fragment. Mapping with additional enzymes has allowed the further ordering of these loci with respect to each other. Together with these data, analysis of a small set of radiation hybrids has suggested the following over-all order of loci within Xq22: centromere-DXS178-DXS94-DXS147-DXS211-DXS17++ +-DXS87- PLP-DXS54-DXS24-DXS83-COL4A5-telomere. The ordering of these random DNA markers, genes, and disease loci, including the genes responsible for Pelizaeus-Merzbacher disease and Alport syndrome, indicates DNA markers that could be of further use clinically for these diseases. Furthermore, this map should form a basis for the refinement of additional disease-associated loci in this region.

Capillary leak after cardiopulmonary bypass operations for correction of congenital heart defects is universally seen in children and often causes significant morbidity and mortality. Since neutrophil-mediated endothelial injury has been implicated as a pathogenetic mechanism, a prospective controlled descriptive study was performed to investigate possible activation pathways during and after the bypass procedure. Eighteen children undergoing operations, nine with cardiopulmonary bypass and nine neurosurgical craniotomy (i.e., operations without bypass), had samples of arterial blood collected at intervals before, during, and after operations. In six of nine cardiac patients circulating interleukin-8 concentrations rose from less than 30 pg/ml to very high concentrations (> 500 pg/ml); in the remaining three patients small rises (peak 57 to 81 pg/ml) were also seen. In all nine, the rise commenced at the time of rewarming, toward the end of bypass, and peaked 1 to 3 hours thereafter. Interleukin-8 release correlated significantly with length of bypass. Interleukin-1 alpha and interleukin-1 beta were not found, and traces of tumor necrosis factor-alpha were detected in one patient only. Circulating elastase alpha 1-antitrypsin concentrations rose simultaneously and correlated significantly with interleukin-8 (p < 0.001) in patients with cardiac disease, as did absolute neutrophil counts (p < 0.001). In contrast, only one of nine patients with neurosurgical disease (undergoing an unusually long operation and exchange transfusion) had a rise in circulating interleukin-8 to levels greater than 500 pg/ml (p < 0.01). The two samples from this patient with elevated interleukin-8 were the only neurosurgical samples with elevated elastase. This study demonstrates the release of interleukin-8 into the circulation after pediatric hypothermic cardiopulmonary bypass and supports the suggestion that this cytokine plays a role in the pathophysiology of capillary leak through neutrophil degranulation.

In 190 pregnancies undergoing cordocentesis for prenatal diagnosis (n = 174) or elective caesarean section (n = 16), fetal peripheral blood B lymphocyte subpopulations were measured using a fluorescence-activated cell sorter (FACScan). The total number of B lymphocytes and polyreactive CD5+ B cells increased exponentially with gestation from respective means of 0.33 x 10(9)/l and 0.25 x 10(9)/l at 17 weeks to a plateau of 0.66 x 10(9)/l and 0.54 x 10(9) at 36 weeks, remaining at that level thereafter. The number of mature CD10- and active CD23+ B lymphocytes increased linearly from a mean of 0.07 x 10(9)/l and 0.11 x 10(9)/l at 17 weeks to 0.24 x 10(9)/l and 0.37 x 10(9)/l, respectively, at 40 weeks. As expected, all B lymphocytes expressed the HLA-DR antigen from as early as 16 weeks gestation. These alterations in specific B lymphocyte subpopulations reflect the pattern of maturation and development of the fetal humoral immune system.

We have previously identified, in three British families having an index child with frequent infections, a point mutation (GGC-->GAC) in codon 54 of exon 1 of the gene for the human lectin mannose binding protein (MBP). This was associated with low serum levels of this complement activating protein and would be anticipated to impair opsonization of mannose rich microorganisms. We now report a second point mutation (GGA-->GAA) in Gambians from West Africa, involving codon 57 of exon 1. By substituting carboxylic acids for axial glycines in the translated proteins both mutations would be expected to disrupt the secondary structure of the collagenous triple helix of the 96 kDa MBP subunits. In the Gambians the codon 57 mutation was studied by PCR, sequence analysis and restriction analysis and found to be remarkably common (frequency of the mutant gene 0.29 in adults and 0.23 in newborns) whereas the codon 54 mutation was very rare (frequency 0.003). However, the codon 54 mutation was frequent in both a British Caucasian and a Hong Kong Chinese population (frequency of the mutant gene 0.17 and 0.11 respectively). It was predicted that both homozygous and heterozygous individuals would have profoundly reduced serum levels of the protein and this was confirmed by immunoassay as was the reduced capacity of such sera to activate complement through the MBP initiated classical pathway. Our data indicate that the two mutations have arisen independently since the divergence of African and non African populations and both have attained high frequencies.