Novel mutations and DNA-based screening in non-Jewish carriers of Tay-Sachs disease

We have evaluated the feasibility of using PCR-based mutation screening for non-Jewish enzyme-defined carriers identified through Tay-Sachs disease-prevention programs. Although Tay-Sachs mutations are rare in the general population, non-Jewish individuals may be screened as spouses of Jewish carriers or as relatives of probands. In order to define a panel of alleles that might account for the majority of mutations in non-Jewish carriers, we investigated 26 independent alleles from 20 obligate carriers and 3 affected individuals. Eighteen alleles were represented by 12 previously identified mutations, 7 that were newly identified, and 1 that remains unidentified. We then investigated 46 enzyme-defined carrier alleles: 19 were pseudodeficiency alleles, and five mutations accounted for 15 other alleles. An eighth new mutation was detected among enzyme-defined carriers. Eleven alleles remain unidentified, despite the testing for 23 alleles. Some may represent false positives for the enzyme test. Our results indicate that predominant mutations, other than the two pseudodeficiency alleles (739C-->T and 745C-->T) and one disease allele (IVS9+1G-->A), do not occur in the general population. This suggests that it is not possible to define a collection of mutations that could identify an overwhelming majority of the alleles in non-Jews who may require Tay-Sachs carrier screening. We conclude that determination of carrier status by DNA analysis alone is inefficient because of the large proportion of rare alleles. Notwithstanding the possibility of false positives inherent to enzyme screening, this method remains an essential component of carrier screening in non-Jews. DNA screening can be best used as an adjunct to enzyme testing to exclude known HEXA pseudodeficiency alleles, the IVS9+1G-->A disease allele, and other mutations relevant to the subject's genetic heritage.     

Unrelated donor bone marrow transplantation for children with acute leukemia

PURPOSE: To test the use of unrelated donor bone marrow transplantation (URD BMT) to cure children with high-risk acute leukemias. PATIENTS AND METHODS: Between June 1985 and December 1994, 50 children with acute leukemia (15 acute myelogenous leukemia [AML], 35 acute lymphoblastic leukemia [ALL]; 22 greater than second complete remission [CR]) received BMT from a URD at the University of Minnesota. Ages ranged from 0.9 to 17.5 years (median, 8.8). Median follow-up is 2.1 years (range, 1 to 7.3). Thirty patients (60%) received bone marrow fully matched at HLA-A,B and DRB1; 20 (40%) received bone marrow with a major or minor mismatch at a single HLA-A or B locus. RESULTS: The median time to neutrophil engraftment was day 24 (range, 14 to 42 days) in those receiving matched and day 25 (range, 15 to 32 days) in those receiving mismatched marrow (P = .35). The incidence of grades III to IV graft-versus-host disease (GVHD) was 23% (95% confidence interval [CI], 7% to 39%) in matched and 32% (95% CI, 8% to 52%) in HLA-mismatched patients (P = .57). The incidence of chronic GVHD was 50% (95% CI, 28% to 72%) in matched and 57% (95% CI, 23% to 91%) in mismatched patients (P = .80). Disease-free survival for patients with ALL is 37% (95% CI, 21% to 53%) at 1 year and 30% (95% CI, 15% to 46%) at 2 years; for patients with AML, 53% (95% CI, 28% to 78%) at 1 year and 33% (95% CI, 6% to 60%) at 2 years. CONCLUSION: URD BMT is an effective treatment for children with poor-prognosis acute leukemia and should be considered for all high-risk patients. Early referral of patients is strongly recommended.     

Coding sequence, exon-intron structure and chromosomal localization of murine TNF-stimulated gene 6 that is specifically expressed by expanding cumulus cell-oocyte complexes

We hypothesized that the direct stimulus of the central chemoreceptor neurons is the CO2/H+-induced change in intracellular pH (pHi). If it is true, pHi responses during hypercapnic stimulation should be exhibited in the central chemoreceptor neurons in the ventral medullary surface (VMS) and some neurons in the CO2/H+ sensitive regions such as the nucleus tractus solitarii of the medial dorsal medulla (MDM). To test this hypothesis, the cultured VMS and MDM neurons (control) derived from one day-old neonate rats were labeled with H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and were exposed to perfusate of various pHs. The H+-sensitive neurons were determined by a rapid decrease in the intracellular BCECF fluorescence intensity. In almost all the MDM neurons (99.6%) and 94% of the VMS neurons, the intracellular BCECF fluorescence intensity remained unchanged when the extracellular pH (pHo) was decreased. In contrast, in 0.4% of the MDM neurons (8/1800) and in 6% of the VMS neurons (111/1800), the intracellular BCECF fluorescence intensity decreased when the pHo was decreased from 7.4 to 7.2. This subpopulation of MDM and VMS neurons were considered to be H+-sensitive neurons. The H+-sensitive neurons in the VMS showed positive immunoreactivity to glutamate (57%, 17/30) and glutamic acid decarboxylase (23%, 7/30), but no immunoreactivity to choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, somatostatin, serotonin and substance P. These results indicate that the H+-sensitive neurons are present specifically in the VMS, and are mainly glutamatergic and GABAergic.     

Small airways dimensions in asthma and in chronic obstructive pulmonary disease

The purpose of this study was to compare the dimensions of the peripheral airways in fatal asthma with those from patients with nonfatal asthma, mild COPD, and normal lung function. Lung specimens from eight individuals who had fatal asthmatic attacks were obtained at postmortem and compared with similar specimens from three asthmatic patients who died of an unrelated cause and four specimens obtained from known asthmatic patients who required lung resection for tumor. These 15 asthmatic lungs were also compared with lungs resected for peripheral neoplasms from 15 patients with normal airway function (FEV1, % of predicted > 85) and 15 patients with mild chronic airflow obstruction (FEV1, % of predicted < 85). All membranous airways with a long-short diameter ratio of 3:1 or less were examined. The smooth muscle and the tissue areas external and internal to the muscle layer were traced using a Bioquant BQ System 4. The same system was used to evaluate the fraction of the submucosa and adventitia taken up by blood vessels. The adventitial, submucosal, and muscle area of the asthmatic airways were greater than those of COPD and control (p < 0.01), and the muscle area was greater in COPD than in control lungs (p < 0.05). These parameters were also greater in the 8 patients with fatal asthma compared with the 7 patients with nonfatal asthma (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic and pharmacodynamic models of the antistaphylococcal effects of meropenem and cloxacillin in vitro and in experimental infection

The efficacies of meropenem (MPM) and cloxacillin (CLC) against two Staphylococcus aureus strains were established in vitro. A pharmacodynamic model equation, based on the concept that the killing rate depends on concentration and time, was fitted to the numbers of CFU. The parameters of the equation are maximum killing rate, time point of maximum killing, and 50% effective concentration (EC50). The EC50s for the two strains were 0.047 and 0.040 mg/liter, respectively, for MPM and 0.105 and 0.121 mg/liter, respectively, for CLC. Calculated values of the parameters were used to predict the numbers of CFU at exponentially decreasing concentrations in vitro as well as in an experimental infection model. The prediction for in vitro conditions gave a satisfactory fit (R2, between 0.862 and 0.894). In vivo the numbers were predicted with the assumption that killing rate in vivo is proportional to that in vitro (R2, between 0.731 and 0.973). The proportionality factor ranged between 0.23 and 0.42; this variation was due mainly to covariation with growth rates in control animals, without other significant differences between antibiotics or strains.     

Contribution of DNA sequence and CAG size to mutation frequencies of intermediate alleles for Huntington disease: evidence from single sperm analyses

New mutations for Huntington disease (HD) arise from intermediate alleles (IAs) with between 29 and 35 CAG repeats that expand on transmission through the paternal germline to 36 CAGs or greater. Using single sperm analysis, we have assessed CAG mutation frequencies for four IAs in families with sporadic HD (IANM) and IAs ascertained from the general population (IAGP) by analyzing 1161 single sperm from three persons. We show that IANM are more unstable than IAGP with identical size and sequence. Furthermore, comparison of different sized IAs and IAs with different sequences between the CAG and the adjacent CCG tracts indicates that DNA sequence is a major influence on CAG stability. These studies provide estimates of the likelihood of expansion of IANM and IAGP to > or = 36 CAG repeats for these individuals. For an IA with a CAG of 35 in this family with sporadic HD, the likelihood for siblings to inherit a recurrent mutation > or = 36 CAG is approximately 10%. For IAGP of a similar size, the risk of inheriting an expanded allele of > or = 36 CAG through the paternal germline is approximately 6%. These risk estimates are higher than previously reported and provide additional information for counselling in these families. Further studies on persons with IAs will be needed to determine whether these results can be generalized to other families.