Novel GAA sequence variant c.1211 A>G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease.

Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO=c.1211A>G and c.1798C>T; AO=c.1211A>G and c.692+5G>T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A>G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A>G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C>T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692+5G>T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A>G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C>T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692+5G>T (vs. c.1798C>T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).

Women's reflections and actions regarding working after breast cancer surgery - a focus group study.

BACKGROUND: To better understand processes affecting return to work (RTW) after breast cancer, more knowledge from the perspective of sickness absentees is warranted. Still, research based on women's own reasoning and actions in RTW is very scarce. This study aims to elucidate how women with breast cancer reflect and act on work-related issues. MATERIAL AND METHODS: Thematic analyses of data from four focus group interviews with 23 women who had had breast cancer surgery in the previous 3-13 months were carried out. RESULTS: The five following themes of reflections regarding RTW were identified: 'health and functioning', 'self-esteem/integrity', 'value of work', 'relationships at work', and 'social circumstances'. These reflections were associated with the three identified themes of actions taken by the women: 'to work or to be sickness absent', 'to adjust work according to own needs or not', and 'to disclose or to hide one's cancer'. There was a distinct difference between women who experienced work as a source of well-being and those who needed a respite from work. CONCLUSION: This study adds knowledge to the process of RTW after breast cancer and focuses on factors that lead the women to an active role in this process. We point to the interplay between women's own preferences, perceived competence, outer opportunities, and the actions each woman take with regard to RTW, which need to be recognized by all stakeholders involved. Furthermore, it continues to be essential to address the specific issue of disclosure in the workplace because this may be distressing for women.

Clinical evaluation of oral methadone in treatment of cancer pain.

A dose-adjustment program for oral methadone and the long-term effects of the analgesic therapy have been evaluated in 15 patients with incurable cancer. Rapid and continuous pain relief without serious side-effects was achieved by "ad libitum" dosage in the first 3-5 days. Thereafter, a dosage based on each patients's subjective need was instituted. The mean daily dose was 44 during the first day and it decreased to 22 mg daily at the end of the dose-adjustment week. Three patients did not complete the program because of insufficient effect or severe nausea. Among the 12 patients who chose to continue the methadone treatment after the initial dose-adjustment period, four continued the therapy to their death, three discontinued the therapy due to insufficient effect, and three due to adverse reactions. In one case it was possible to stop the treatment due to decreased pain. The treatment period in these 12 patients varied between 8 and 270 days. Oral methadone offers good pain relief for long periods of time in this group of patients and has obvious advantages as compared to long term parenteral therapy with narcotic analgesics.

Clinical pharmacokinetics of methadone.

Studies with single doses of methadone have shown that the oral biological availability is 79 +/- 21%, range 41-99%. The rate of elimination is mostly due to metabolic clearance. Below a urinary pH of 6, renal clearance becomes of quantitative importance. In five subjects treated with ammonium chloride (acidic urine), the plasma half-life of methadone was found to be 19.5 +/- 3.6 h. When treated with sodium bicarbonate the same subjects had plasma half-lives of 42.1 +/- 8.8 h. During continuous treatment with methadone, cellular tolerance may occur and in some subjects also metabolic tolerance. In treatment of severe cancer pain such adaptive changes in methadone pharmacodynamics and pharmacokinetics are best managed by a regimen involving a fixed dose but a flexible and patient-controlled dosage interval.

Patient-controlled dose regimen of methadone for chronic cancer pain.

Fourteen patients with severe cancer pain participated in a trial of methadone given in a fixed dose (10 mg) but at intervals selected by the patients themselves during the loading phase. The aim was to achieve rapid pain relief while avoiding the risk of toxicity from accumulation of methadone. As expected, the dosage intervals increased gradually over the first few days of treatment, the daily dose decreasing from 30-80 mg on the first day to 10-40 mg at the end of the week. Plasma concentrations of methadone varied sevenfold after four to five days (0.24 to 1.75 mumol/1; 7.4 to 54.2 microgram/100 ml). Eleven patients reported complete or almost complete pain relief and elected to continue with methadone after the study. In no case was treatment withdrawn because of intoxication. From these findings a patient-controlled dosage regimen of oral methadone may be an effective and safe alternative to parenteral narcotic medication, adjusting both for individual variation in pain intensity and for pharmacokinetics.

Pharmacokinetics of methadone during maintenance therapy: pulse labeling with deuterated methadone in the steady state.

A technique is presented for study of steady state kinetics of methadone using pulse labeling with deuterated methadone (d3) and mass fragmentography to measure both unlabeled and labeled methadone in blood. Seven subjects maintained on methadone for at least 10 months were admitted to a closed metabolic ward. The daily dose of unlabeled methadone (d0) was substituted by one dose of methoadone-d3 and plasma levels of methadone-d0 and methadone-d3 were followed for 48 h using a precise (SD +/- 5%) and sensitive (30 pmol/ml) mass fragmentographic technique. Plasma half-lives (T1/2) for both methadone-do and methadone-d3 were calculated from samples obtained 8--24 H following the dose of methadone-d3. The t1/2 of oral methadone-d3 was shorter (22 +/- 2 h) than that of methadone-d0 (52 +/- 20 h). The same pattern was observed after intravenous administration. The results indicate multiple pools of methadone in the body.