Ataxia-telangiectasia: phenotype/genotype studies of ATM protein expression, mutations, and radiosensitivity.

Previous studies on a limited number of ataxia-telangiectasia (A-T) patients with detectable levels of intracellular ATM protein have suggested a genotype/phenotype correlation. We sought to elucidate this possible correlation by comparing ATM protein levels with mutation types, radiosensitivity, and clinical phenotype. In this study, Western blot analysis was used to measure ATM protein in lysates of lymphoblastoid cell lines (LCLs) from 123 unrelated A-T patients, 10 A-T heterozygotes, and 10 patients with phenotypes similar to A-T. Our Western blot protocol can detect the presence of ATM protein in as little as 1 microg of total protein; at least 25 microg of protein was tested for each individual. ATM protein was absent in 105 of the 123 patients (85%); most of these patients had truncating mutations. The remaining subset of 18 patients (15%) had reduced levels of normal-sized ATM protein; missense mutations were more common in this subset. We used a colony survival assay to characterize the phenotypic response of the LCLs to radiation exposure; patients with or without detectable ATM protein were typically radiosensitive. Nine of 10 A-T heterozygotes also had reduced expression of ATM, indicating that both alleles contribute to ATM protein production. These data suggest that although ATM-specific mRNA is abundant in A-T cells, the abnormal ATM protein is unstable and is quickly targeted for degradation. We found little correlation between level of ATM protein and the type of underlying mutation, the clinical phenotype, or the radiophenotype.

Ataxia-telangiectasia: identification and detection of founder-effect mutations in the ATM gene in ethnic populations.

To facilitate the evaluation of ATM heterozygotes for susceptibility to other diseases, such as breast cancer, we have attempted to define the most common mutations and their frequencies in ataxia-telangiectasia (A-T) homozygotes from 10 ethnic populations. Both genomic mutations and their effects on cDNA were characterized. Protein-truncation testing of the entire ATM cDNA detected 92 (66%) truncating mutations in 140 mutant alleles screened. The haplotyping of patients with identical mutations indicates that almost all of these represent common ancestry and that very few spontaneously recurring ATM mutations exist. Assays requiring minimal amounts of genomic DNA were designed to allow rapid screening for common ethnic mutations. These rapid assays detected mutations in 76% of Costa Rican patients (3), 50% of Norwegian patients (1), 25% of Polish patients (4), and 14% of Italian patients (1), as well as in patients of Amish/Mennonite and Irish English backgrounds. Additional mutations were observed in Japanese, Utah Mormon, and African American patients. These assays should facilitate screening for A-T heterozygotes in the populations studied.

Linkage studies exclude the AT-V gene(s) from the translocation breakpoints in an AT-V patient.

An 8-year-old girl with severe microcephaly of prenatal onset, borderline intelligence, defects of skin pigmentation, deficiency of both humoral and cellular immunity, a normal serum alpha-fetoprotein level and hypersensitivity to ionizing irradiation is described. Spontaneous chromosomal breakage in lymphocytes together with the clinical presentation led to the diagnosis of ataxia telangiectasia variant (AT-V). In addition, the patient carried a constitutional translocation of paternal origin: 46,XX,t(3;7)(q12;q31.3) pat. In subsequent linkage and haplotype studies in 12 AT-V families with microsatellite markers from each of the translocation breakpoint regions, we could clearly exclude the localization of an AT-V gene to these regions.

Ataxia-telangiectasia: mutations in ATM cDNA detected by protein-truncation screening.

We have examined the distal half of the ataxia-telangiectasia (A-T) gene transcript for truncation mutations in 48 A-T affecteds. We found 21 mutations; 4 of the mutations were seen in more than one individual. Genotyping of the individuals sharing mutations, by using nearby microsatellite markers, established that three of the four groups shared common haplotypes, indicating that these were probably founder effects, not public mutations. The one public mutation was found in two American families, one of Ashkenazi Jewish background and the other not. Most truncations deleted the PI3-kinase domain, although some exceptions to this were found in patients with typical A-T phenotypes. All patients not previously known to be consanguineous were found to be compound heterozygotes when mutations could be identified--that is, normal and abnormal protein segments were seen on SDS-PAGE gels. All 48 patients gave RT-PCR products, indicating the presence of relatively stable mRNAs despite their mutations. These results suggest that few public mutations or hot spots can be expected in the A-T gene and that epidemiological studies of A-T carrier status and associated health risks will have to be designed around populations with frequent founder-effect mutations, despite the obvious limitations of this approach.

Identification of Btk mutations in 20 unrelated patients with X-linked agammaglobulinaemia (XLA).

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency resulting from mutations in the gene for a cytoplasmic protein tyrosine kinase (Btk). We have utilised reverse-transcription-based PCR in combination with the chemical cleavage and mismatch technique (CCM) to screen for Btk mutations in 42 unrelated patients having classical XLA or 'leaky' XLA-like phenotypes. A variety of mutations, including point mutations, large deletions and splicing defects were detected using this strategy. In total, 20 mutations were found in these patients. All the mutations were different with the exception of three unrelated patients who all showed the same Arg-->His amino acid substitution (R641H) at a highly-conserved residue in the kinase domain. We have also used structural modelling of the Btk kinase domain to predict how two different amino acid substitution mutations at highly-conserved residues are likely to affect the Btk kinase activity.

Clinical efficacy of intravenous immunoglobulin in patients with severe inflammatory chest disease and IgG3 subclass deficiency.

To investigate the efficacy of i.v. IgG treatment in pediatric patients with inflammatory lung disease, a prospective, controlled clinical trial was carried out over a 2-year study period. Patients were enrolled on the basis of severe clinical symptomatology. After 1 year of conventional treatment, the patients received 400 mg/kg per month of an i.v. IgG product containing only trace amounts of IgG3 in addition to their regular treatment throughout the second year. Significant clinical improvement, as documented by duration of hospital stay (first year 27.8 days, second year 4.9 days), use of antibiotics (132.8 versus 30.9 days) and use of steroids (21.4 versus 0.7 days) could be observed. Data obtained on a subgroup of patients with IgG3 deficiency were analysed separately. These results indicate that patients with severe chest disease who have IgG3 deficiency will also benefit from i.v. IgG treatment. The mode of action cannot be attributed to replacement of the respective isotypes, but is probably due to the effect of i.v. IgG in preventing repeated viral infections.

IgG subclasses and antibody response to pneumococcal capsular polysaccharides in children with severe sinopulmonary infections and asthma.

A prospective open study was carried out on 30 pediatric patients with most severe chest disease whose serum immunoglobulin levels were normal. The patients entered into the study had had two or more documented episodes of pneumonia, and/or six episodes of bronchitis with fever within a year, and/or severe asthma (steroid-dependent), and/or hospitalization for chest disease for more than 30 days within the year preceding the study. Eleven patients had sinopulmonary infections, 19 had asthma. Twenty patients had low levels of one or two IgG subclasses: 11 were deficient in IgG3, three in IgG4, three in IgG3 + IgG4, and three in IgG2 + IgG4. Patients with low IgG subclass levels were distributed throughout the different clinical entities. These children had significantly longer periods of hospitalization than the patients in whom all IgG subclasses were within the normal range. They suffered more often from sinopulmonary infections. Asthmatic children with low levels of an IgG subclass reported more days with wheezing and needed more steroids than the children without subclass deficiencies.