Roland J. Levinsky

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector

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have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two.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.

Molecular basis of opsonic defect in immunodeficient children

Low serum mannose-binding protein (MBP) concentrations are associated with a common opsonic defect. Sequence analysis of the MBP gene in three children with recurrent infections, the opsonic defect, and low serum MBP concentrations showed a point mutation at base 230 of exon 1 causing a change of codon 54 from GGC to GAC. The replacement of glycine with an aspartic acid residue disrupts the fifth Gly-Xaa-Yaa repeat in the collagen-like domain of each 32 kD MBP peptide chain and probably prevents the formation of the normal triple helix. Study of sixteen members of the three families showed autosomal dominant co-inheritance of the mutation and low serum MBP concentrations.

High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene.

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.

Preparative procedures of cooling and re-warming increase leukocyte integrin expression and function on neotrophils

The majority of studies involving neutrophil integrin expression and function are performed at physiological temperatures subsequent to routine preparative procedures at 4 degrees C. We have shown that surface expression of the leukocyte integrin molecules on neutrophils is increased by cooling and subsequently re-warming of neutrophils to 37 degrees C when compared with cells held at room temperature or 37 degrees C. This increase in expression is secondary to prior cooling of the neutrophils. There is an associated increase in function of these newly expressed adhesion molecules, making the neutrophils more adherent to endothelium. Preparation of cells at 4 degrees C and subsequently warmed to 37 degrees C is stimulatory for neutrophils, probably causing translocation of intracellular stores of the leukocyte integrins to the cell surface in a manner analogous to the stimulant FMLP. Our results indicate that the cooling of neutrophils during isolation is an inappropriate method of neutrophil preparation.

Prognosis of chronic granulomatous disease.

The records of 28 patients with chronic granulomatous disease born over a 32 year period were reviewed. The characteristics of the group, and the frequency with which various clinical and laboratory features had been recorded, was assessed. Nine patients were known to have died, in most cases of progressive suppurative infection. Actuarial analysis showed 50% survival through the third decade of life. The long term survival of patients developing symptoms after the end of the first year of life was significantly better than that of patients whose illness started in infancy. Our data confirm that the severity of chronic granulomatous disease is not uniform, and that the prognosis for long term survival is better than that suggested in earlier reports. Early onset may be a poor prognostic sign and invasive aspergillosis is a life threatening complication. In the absence of curative treatment, trials to assess the effectiveness of interferon gamma are necessary and early antenatal diagnosis should be offered to as many affected families as possible.

High-Titer Recombinant Adeno-Associated Virus Production from Replicating Amplicons and Herpes Vectors Deleted for Glycoprotein H

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.

Identical point mutation leading to low levels of mannose binding protein and poor C3b mediated opsonisation in Chinese and Caucasian populations

A common opsonic defect occurring in 7% of the Caucasian population is associated with low serum levels of the lectin mannose binding protein (MBP). This study sought to determine whether the deficiency was also present in a Chinese population using sera obtained from 100 healthy Chinese children (age range 6 weeks-16 years). The distribution profiles of MBP levels and C3b/C3bi fragments binding to mannan coated plates were both bimodal and similar to the corresponding Caucasian profiles. Serum MBP levels were low in 9% of the Chinese children and all of these sera generated low levels of C3b/C3bi fragments. Overall there was a high significant correlation between MBP levels and C3b opsonin generation (r = 0.77; P less than 0.001). By analogy with similar findings in a Caucasian population we believe this correlation to be a reflection of antibody independent complement activation by MBP. In a pilot study of DNA obtained from three adult Chinese with low MBP levels the point mutation causing MBP deficiency in Caucasians was identified in all three cases.

Use of X chromosome inactivation analysis to establish carrier status for X-linked severe combined immunodeficiency.

Analysis of X chromosome inactivation in T-lymphocyte DNA from two obligate carriers of X-linked severe combined immunodeficiency showed a non-random pattern. This method was then used to establish carrier status in at-risk females in X-linked pedigrees. It was further used to differentiate between X-linked and autosomal recessive inheritance of the disease when the mode of inheritance was not clear from the pedigree. In addition, a mother of a boy affected by the sporadic form of the disease was found to have non-random X inactivation in her T lymphocytes and she is therefore a carrier of the X-linked disease.

A Defined Window for Efficient Gene Marking of Severe Combined Immunodeficient-Repopulating Cells Using a Gibbon Ape Leukemia Virus-Pseudotyped Retroviral Vector

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.