Identification of new Fas mutations in a patient with autoimmune lymphoproliferative syndrome (ALPS) and eosinophilia.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare, newly recognized, chronic lymphoproliferative disorder in children and is characterized by lymphadenopathy, splenomegaly, pancytopenia, autoimmune phenomena and expansion of double-negative (DN) T lymphocytes (TCR alpha beta+, CD4-, CD8-). Defective lymphocyte apoptosis caused by mutations of the Fas (CD95) gene has been linked in the pathogenesis of ALPS, as binding of Fas-ligand to Fas can trigger apoptosis. Of the ALPS cases reported to date, point mutations, frameshifts and silent mutations in Fas all have been identified. We report two new point mutations in Fas in a child with ALPS and eosinophilia; studies on other family members established the pattern of inheritance for these mutations. Flow cytometric analysis of blood and tissues (spleen, lymph node, bone marrow) revealed abnormally expanded populations of DN T lymphocytes. Furthermore, activated lymphocytes and IFN gamma-activated eosinophils were resistant to Fas-mediated apoptosis. Eosinophil resistance to Fas-mediated apoptosis has not been previously described in ALPS. Sequencing of Fas revealed two separate mutations not previously reported. One mutation, a C to T change at base 836, was a silent mutation inherited from the mother, while the second mutation, a C to A change at base 916, caused a non-conservative amino acid substitution in the death domain of Fas, changing a threonine to a lysine. This mutation is associated with a predicted change in the structure of a part of the death domain from a beta-pleated sheet to an alpha-helix. We speculate that the mutation in the death domain prevents the interaction of Fas with intracellular mediators of apoptosis and is responsible for the autoimmune manifestations of ALPS and the abnormal lymphocytosis and eosinophilia in this patient.

Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction. A multicenter study.

OBJECTIVE--To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR). DESIGN--DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive. SETTING--Fourteen university-affiliated and private genetic disease diagnostic laboratories. PATIENTS--Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene. MAIN OUTCOME MEASURES--The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored. RESULTS--The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%. CONCLUSIONS--Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.

The use of DNA probes to establish parental origin in Down syndrome.

Molecular analysis was performed to determine the parental origin of the extra number 21 chromosome in 20 couples following the birth of a child with standard trisomy 21. The parent of origin was successfully identified in 9 of 20 (45%) using five chromosome-21-specific DNA probes and eight restriction endonucleases by restriction fragment length polymorphism and dosage analysis; seven were of maternal and two of paternal origin. Utilizing the observed allele frequencies, the expected frequencies of informative homozygous matings [2(p2q2)] approximate 10% for seven of eight enzyme/probe combinations; the eighth, TaqI/pPW231F (D21S3), is 3%. The observed phenotype frequencies for all enzyme/probe combinations tested conform closely to predictions by the Hardy-Weinberg law. Strong linkage disequilibrium was observed between the DNA markers of EcoRI and TaqI with probe pPW236B; identical results were obtained with G-95 alpha 1-11a. We were able to demonstrate that although these two probes are of different size, and hence not identical, they detect the same TaqI and EcoRI polymorphisms; therefore both should be assigned to a single locus, D21S11.

Micronucleus assay in human fibroblasts: a measure of spontaneous chromosomal instability and mutagen hypersensitivity.

By comparing fibroblast strains derived from individuals exhibiting chromosome instability and/or mutagen hypersensitivity (Cockayne syndrome, ataxia telangiectasia, and Fanconi anemia) with strains derived from healthy donors, the fibroblast micronucleus assay has been established as a reproducible measure of the genotypic variation in spontaneous or mitomycin C (MMC)-induced chromosomal instability. The patient strains that were moderately or exquisitely sensitive to MMC, whereas the mildly sensitive strain (Cockayne syndrome) overlapped with the control range. The reproducibility of the assay was evaluated within and between experiments. Paired comparison analyses between duplicate cultures and between repeat experiments failed to show any significant differences between micronucleus frequencies within strains, whereas a significant differences in the spontaneous micronucleus frequencies between strains was observed. In addition to its value as a test system for genotoxins, the fibroblast micronucleus assay may be useful for investigating genetically determined hypersensitivity to mutagens, elevated spontaneous chromosomal breakage, and chromosome segregation errors.

Hemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes.

We have used two strategies to study 14 hemophilia B families from 11 kindreds for possible carrier detection and prenatal diagnosis. First, we sequentially used the Factor IX probes (sequentially with restriction enzymes Taq I, Xmn I, and Dde I), and the linked probes p45h (Taq I), p45d (Pst I), and 52a (Taq I) for restriction fragment length polymorphism (RFLP) analysis. Second, we searched for useful variant Taq I digestion fragments using the Factor IX complementary DNA. Two separate new Taq I variants in exon VIII were identified. Using both strategies, 11 of 14 families (from 9 of 11 kindreds) were informative for further studies. In five kindreds studied in detail, the carrier status of all 11 at risk females was determined and prenatal diagnosis could be offered to the offsprings of each of the six carriers identified. Thus, in this study, we have identified a higher proportion of informative families than has previously been reported.

Induction of mitochondrial mutations in human cells by methotrexate.

Inhibition of dihydrofolate reductase by the folate analog, methotrexate (MTX) results in a depletion of tetrahydrofolate dependent one carbon transfer reactions in amino acid and nucleic acid biosynthesis. When human cells (either HeLa or normal skin fibroblasts) are exposed to MTX in a defined medium containing dialyzed fetal calf serum, essential and non-essential amino acids, and purine source, the thymidylate pools alone are depleted. Under these conditions exposure to 10(-6) M MTX induces mitochondrial mutagenesis, measured as an increase in the frequency of chloramphenicol resistant (CAPR) colonies, without altering the rate of nuclear mutation monitored by determining the frequency of 6-thioguanine resistance (TGr). The occurrence of CAPR mutations is time, and MTX concentration dependent and the frequency of CAPR can be decreased quantitatively by adding thymidine to the culture medium. This mitochondrial specific mutagenesis can also be achieved using the thymidylate synthetase inhibitor, 5-fluorodeoxyuridine further implicating thymidylate pools as the mediator of this effect. During the course of exposure to 10(-6) M MTX the thymidine kinase deficient HeLa BU25 cell line exhibits a progressive depletion and degradation of mitochondrial DNA suggesting that the mutagenesis and DNA degradation represent portions of a progressive process. The basis for the selective sensitivity of the mitochondrial genome to thymidylate depletion mutagenesis may be the consequence of its differences from the nuclear genome in mechanisms of DNA replication or repair.

Factors distinguishing couples at risk for nondisjunction.

Micronucleus frequencies were determined on 24 young parents of trisomic infants, 21 individuals with recurrent unexplained abortions, and 42 control individuals with proven reproductive success. In addition to measurements of spontaneous micronucleus frequencies, mitomycin C induced frequencies were determined at two doses (2.5 and 5.0 ng/mL). Using the 95% confidence limits established from control data as an arbitrary upper limit, 16 of 24 parents of trisomics and 5 of 21 recurrent aborters were detected by the micronucleus assay to above this cutoff. The effects of sex, age, pregnancy status, and a variety of environmental exposures were studied by comparing the micronucleus frequencies of the exposed and unexposed populations. The data suggested that vitamins were associated with a lower micronucleus frequency and tea drinking with an increased micronucleus frequency in parents of trisomics, an effect not seen in controls. These results suggest that a biologic basis for nondisjunction may be associated with elevation in spontaneous and induced micronuclei. In addition, tea and vitamins may modulate micronucleus frequencies in parents of trisomics who appear to be more sensitive to these influences than the controls.