Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and Becker muscular dystrophy.

BACKGROUND: Recent methodological advances have improved the detection rate for dystrophin mutations, but there are no published studies that have measured the clinical utility of these protocols for carrier detection compared with conventional carrier testing protocols that use pedigree, serum creatine kinase levels and linkage analysis. METHODS AND SUBJECTS: The clinical utility of a combined mutation detection protocol was measured. It involved quantitative PCR procedures followed by DNA sequence analysis for the identification of dystrophin mutation carriers in 2101 women at risk of being carriers from 348 mutation-known Duchenne or Becker muscular dystrophy pedigrees. RESULTS: The combined mutation detection protocol identified a mutation in 96% and 82% of index cases of Duchenne muscular dystrophy and Becker muscular dystrophy, respectively. An additional 692 (33%) potential carriers were correctly classified by the combined mutation detection protocol compared with pedigree, serum creatine kinase levels and linkage analysis. Significantly lower mutation carrier rates were identified in the mothers of isolated cases with deletion mutations than predicted from theoretical considerations, but these findings were not confirmed for duplication and DNA sequence mutations. CONCLUSIONS: There are significant clinical benefits to be gained from a combined mutation detection protocol for carrier detection. It is recommended that mutation-specific carrier frequencies for the different classes of dystrophin mutations should be taken into account in genetic counselling practice.

Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy.

BACKGROUND: A significant component of the variation in cognitive disability that is observed in Duchenne muscular dystrophy (DMD) is known to be under genetic regulation. In this study we report correlations between standardised measures of intelligence and mutational class, mutation size, mutation location and the involvement of dystrophin isoforms. METHODS AND RESULTS: Sixty two male subjects were recruited as part of a study of the cognitive spectrum in boys with DMD conducted at the Sydney Children's Hospital (SCH). All 62 children received neuropsychological testing from a single clinical psychologist and had a defined dystrophin gene (DMD) mutation; including DMD gene deletions, duplications and DNA point mutations. Full Scale Intelligence Quotients (FSIQ) in unrelated subjects with the same mutation were found to be highly correlated (r = 0.83, p = 0.0008), in contrast to results in previous publications. In 58 cases (94%) it was possible to definitively assign a mutation as affecting one or more dystrophin isoforms. A strong association between the risk of cognitive disability and the involvement of groups of DMD isoforms was found. In particular, improvements in the correlation of FSIQ with mutation location were identified when a new classification system for mutations affecting the Dp140 isoform was implemented. SIGNIFICANCE: These data represent one of the largest studies of FSIQ and mutational data in DMD patients and is among the first to report on a DMD cohort which has had both comprehensive mutational analysis and FSIQ testing through a single referral centre. The correlation between FSIQ results with the location of the dystrophin gene mutation suggests that the risk of cognitive deficit is a result of the cumulative loss of central nervous system (CNS) expressed dystrophin isoforms, and that correct classification of isoform involvement results in improved estimates of risk.

Osmoregulation in the parasitic protozoan Tritrichomonas foetus.

Tritrichomonas foetus was shown to undergo a regulatory volume increase (RVI) when it was subjected to hyperosmotic challenge, but there was no regulatory volume decrease after hypoosmotic challenge, as determined by using both light-scattering methods and measurement of intracellular water space to monitor cell volume. An investigation of T. foetus intracellular amino acids revealed a pool size (65 mM) that was similar to that of Trichomonas vaginalis but was considerably smaller than those of Giardia intestinalis and Crithidia luciliae. Changes in amino acid concentrations in response to hyperosmotic challenge were found to account for only 18% of the T. foetus RVI. The T. foetus intracellular sodium and potassium concentrations were determined to be 35 and 119 mM, respectively. The intracellular K(+) concentration was found to increase considerably during exposure to hyperosmotic stress, and, assuming that there was a monovalent accompanying anion, this increase was estimated to account for 87% of the RVI. By using light scattering it was determined that the T. foetus RVI was enhanced by elevated external K(+) concentrations and was inhibited when K(+) and/or Cl(-) was absent from the medium. The results suggested that the well-documented Na(+)-K(+)-2Cl(-) cotransport system was responsible for the K(+) influx activated during the RVI. However, inhibitors of Na(+)-K(+)-2Cl(-) cotransport in other systems, such as quinine, ouabain, furosemide, and bumetanide, had no effect on the RVI or K(+) influx in T. foetus.

Menadione kills trophozoites and cysts of Giardia intestinalis.

Production of reactive oxygen species by redox cycling in the presence of low levels of oxygen has been studied as a possible approach to anti-protozoal chemotherapeutic strategy. Incubation of the diplomonad flagellate Giardia intestinalis with 2-methy-1,4-naphthoquinone (menadione), under anaerobic conditions, gave UV absorption changes characteristic of reduction to menadiol; partial reversal was observed on admitting O(2). Under microaerobic conditions, similar to those on the surface of the jejunal mucosa, trophozoites consumed O(2) rapidly in the presence of menadione; reaction products included singlet O(2) (monitored by single photon counting of O(2)-dependent low-level chemiluminescence) and H(2)O(2) (measured by the formation of Complex I of microperoxidase). Trophozoites became swollen and incapable of regulatory volume control; these irreversible responses led to loss of motility, cessation of flagellar activity and cell death. Comparison of the sensitivities of trophozoites to metronidazole and menadione gave LC(50) values ( microg x ml(-1)) of 1.2 and 0.7, respectively; corresponding values for cysts (measured by in vitro excystation capacities) were >50 and 1.3. Menadione (LD(50) in mice, 0.5 g kg(-1)) is therefore a potentially more useful and general anti-giardial agent than metronidazole, as it is active against cysts as well as trophozoites.

The "primitive" microaerophile Giardia intestinalis (syn. lamblia, duodenalis) has specialized membranes with electron transport and membrane-potential-generating functions.

Here it is shown that the flagellated protozoon Giardia intestinalis, commonly regarded as an early branching eukaryote because of its lack of mitochondria, has membraneous structures that partition the cationic, membrane-potential-sensitive fluorophore rhodamine 123. This organism also reduces a tetrazolium fluorogen at discrete plasma-membrane-associated sites. That these functions occur in distinctive specialized membrane systems supports the growing evidence that G. intestinalis may not be primitive, but is derived from an aerobic, mitochondria-containing flagellate.

The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity.

Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O(2). Thus, when exposed to controlled concentrations of dissolved O(2) >8 microM, they gradually lose their ability to scavenge O(2). In a washed cell suspension stirred under 10% air in N(2) (equivalent to 25 microM O(2)), inactivation of the O(2)-consuming system was complete after 3.5 h; during this period accumulation of H(2)O(2) (3 micromol per 10(6) organisms) and oxidation of cellular thiols to 16% of their initial level occurred. Under 20% air (50 microM O(2)), respiratory inactivation was complete after 1.5 h, and under air (258 microM O(2)), after 50 min. Loss of O(2)-consuming capacity was accompanied by loss of motility. Use of the fluorogen 2, 7-dichlorodihydrofluorescein acetate indicated that intracellular H(2)O(2) is produced at extranuclear sites. Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC(4)(3), showed that values declined from -134 mV to -20 mV after 4.5 h aeration. Incubation of organisms with 60 microM H(2)O(2) for 10 min gave partial collapse of plasma membrane potential and complete loss of O(2) uptake capacity; motility and viability as assessed by DiBAC(4)(3) exclusion were completely lost after 1 h. Inactivation of the O(2)-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.