Reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells. II. Enhancement of the alkylating activity of AZQ by NAD(P)H: quinone-acceptor oxidoreductase (DT-diaphorase).

The alkylating activity of reduced diaziquone was studied by the nitrobenzylpyridine (NBP) assay and was compared to those of the parent compound and aziridine-containing N,N',N"-triethylenethiophosphoramide (Thio-TEPA). Diaziquone (AZQ) was reduced enzymatically by 2e- using S9 cell fraction from MCF-7 cells which is rich in NAA(P)H:quinone-acceptor oxidoreductase (DT-diaphorase) (QAO) activity. One electron enzymatic reduction was performed with NADPH-cytochrome c reductase. The alkylating activity of AZQ increased 3-fold when reduced by 2e-. This increase was inhibited by dicumarol, an inhibitor of QAO. In contrast, the alkylating activity of AZQ did not increase beyond that of the parent compound when reduced by 1e- using purified NADPH-cytochrome c reductase. Similar results were obtained when AZQ was reduced chemically with borohydride (2e-) and with NADPH (1e-). Anaerobic incubations of AZQ with the S9 fraction of MCF-7 cells (2e- reduction) resulted in an increase in NBP alkylation over its aerobic counterpart (1.8-fold) while maintaining the near 3-fold increase in alkylation over untreated AZQ. In contrast, AZQ incubations with NADPH-cytochrome c reductase (1e- reduction) under the same conditions did not result in an NBP alkylation increase over untreated AZQ. These results indicate that AZQ hydroquinone is most likely the responsible species for the observed alkylation of this antitumor agent to DNA and other nucleophiles. The results also suggest that NAD(P)H:quinone-acceptor oxidoreductase is a very important enzyme in the bioactivation of AZQ.


GEA 857 blocks potassium channels in the membrane and, thereby, prolongs muscarinic cholinergic responses in N1E-115 neuroblastoma cells.

GEA 857 [2-(4-chlorophenyl)-1,1-dimethylethyl 2-amino-3-methylbutanoate], a structural analogue of the serotonin (5-HT) uptake inhibitor alaprocalate but without effects on the 5-HT uptake, was shown to potentiate muscarinic cholinergic responses in N1E-115 neuroblastoma cells. In intracellular recording experiments, GEA 857 (1 microM) increased the cell input resistance and prolonged the action potential. It also prolonged the cellular response to carbachol acting on muscarinic receptors in a manner mimicked by potassium channel blockers such as 4-aminopyridine and TEA. GEA 857 did not affect the carbachol stimulated uptake of 45Ca, but depressed the carbachol activated outflow of 86Rb from neuroblastoma cells. The conclusion drawn from these results is that GEA 857 reduces potassium conductances in the membrane in N1E-115 neuroblastoma cells and, thereby, prolongs muscarinic agonist-induced responses.


Induction, purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus).

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatography, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a M(r) of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.


Antiplatelet activity of the long-acting thromboxane receptor antagonist BMS 180,291 in monkeys.

The effects of the novel TxA2/prostaglandin endoperoxide (TP) receptor antagonist BMS 180,291 on platelet reactivity was determined ex vivo in conscious African green monkeys. Platelet aggregation responses to U-46,619 were decreased 50% and 100% at 23 to 24 hrs after BMS 180,291 oral doses of 1 and 3 mg/kg, respectively. In addition to inhibiting aggregation, a 3 mg/kg oral dose of BMS 180,291 also produced an 11 +/- 3-fold shift to the right in the U-46,619 concentration-response relationship for platelet shape change at 24 hrs after dosing. When the 3 mg/kg oral dose was continued for 11 days, the shift in this concentration-response relationship increased to 26 +/- 10- and 93 +/- 30-fold at 24 hrs after the 8th and 11th doses, respectively. This progressive inhibition corresponds to 93 +/- 3 and 99 +/- 1% blockade of platelet TP-receptors responsible for shape change, respectively. Comparable levels of TP-receptor blockade have been previously correlated with antithrombotic and antiischemic activities of TP-receptor antagonists in vivo. Platelet reactivity to U-46,619 had completely recovered on the 7th day after the final dose of BMS 180,291, indicating effective elimination from the circulation over this interval. In separate experiments, a 3-mg/kg i.v. dose of BMS 180,291 produced only marginal and transient hemodynamic effects in anesthetized African green monkeys. Overall, these data demonstrate that BMS 180,291 given orally once a day produces a sustained and therapeutically-relevant level of TP-receptor antagonism.


The relationship between spontaneous coping strategies and perceived anxiety of patients undergoing cardiac catheterization.

Little is known about how patients spontaneously cope with the inherent stressors of the cardiac catheterization procedure. In this study, spontaneous coping strategies were identified and the relationship between coping strategies and anxiety throughout the procedure was examined. Data consisted of 62 patients' subjective ratings of anxiety, reported at 6 points during the procedure, and the coping strategies which were used at those times. The Coping with Invasive Medical Procedures (CIMP) Scoring System was developed and utilized to examine the relationships between coping and anxiety. At various points throughout the procedure, "control" and "hoping" were associated with high anxiety, while "passive coping" and "positive reframing" were associated with low anxiety. Patients who repeatedly changed coping strategies during the procedure experienced slightly less anxiety than those using a more consistent coping approach. The results are discussed with an emphasis on patient teaching.


Discharge functional capacity and self-efficacy of men after coronary artery bypass graft surgery.

Fatigue and activity intolerance are reported to persist for several weeks after discharge after coronary artery bypass graft (CABG) surgery. This may be due to early discharge, which limits the time, prior to leaving hospital, to achieve an adequate functional level for the performance of many activities of daily living. Alternatively, it may be related to level of self-confidence, or self-efficacy (SE), in one's ability to perform physical activity. High SE may result in overexertion during the vulnerable, early post-discharge phase of recovery, whereas low SE may cause underexertion. The limited number of cardiac rehabilitation programs in Canada for this phase of recovery precludes close guidance and monitoring during physical activity for most patients. Prior to prescribing safe and effective home exercise at discharge, it is essential to assess functional level, cardiovascular responses to physical activity, and perceived level of confidence in ability to perform physical activity. The purpose of this descriptive study was to measure functional capacity, hemodynamic responses to low level exercise, and self-efficacy at discharge after CABG surgery. Twenty-one men completed a self-efficacy questionnaire (SEQ) and a low-level graded exercise test (LL-GXT) using a modified Naughton protocol, on the day of discharge. Results revealed that discharge functional capacity ranged from 1.0 METs to 4.3 METs, peak heart rate ranged from 82-150 beats.min-1, and peak systolic blood pressure from 114-200 mmHg. Subjects were more confident in their ability to tolerate psychological stressors than physical activity. Furthermore, there was no correlation between SE for physical activity and the physiological variables except HR.(ABSTRACT TRUNCATED AT 250 WORDS)


Xenopus Gastrulation without a blastocoel roof.

The objective of this paper is to determine the function in gastrulation of several of the five major regional morphogenetic processes in the African clawed frog, Xenopus laevis. These regional processes are (1) epiboly of the animal cap (AC); (2) migration of the mesoderm on the roof of the blastocoel; (3) convergence and extension of the dorsal, noninvoluting marginal zone (NIMZ); (4) formation of the bottle cells at the site of blastopore formation; and (5) convergence and extension of the involuting marginal zone (IMZ). After the AC and the NIMZ were removed, thus eliminating the first three of these processes, the IMZ involuted, constricted, and closed the blastopore. It also converged and extended to form notochord and somites, although these tissues were often crooked and sank into or were covered over by the vegetal endoderm. When the AC was removed, the dorsal axial mesoderm involuted and stuck to the inner surface of the NIMZ. The IMZ and NIMZ converged and extended together to form a longer, straighter axis than that formed by the IMZ alone. Moreover, presence of the NIMZ also prevented the sinking of the IMZ into the endoderm. Misalignment of the available AC-NIMZ substratum and the IMZ at the beginning of gastrulation suggested that the IMZ determines the general direction of its own extension. Absence of the AC-NIMZ accelerated and increased the normal effects of bottle cell formation on the IMZ and vegetal endoderm. In absence of the AC-NIMZ as a substratum on which to migrate, prechordal mesoderm was pushed anteriorly by the converging and extending mesoderm behind it, but it did not spread normally. We conclude that (1) involution and blastopore closure by the IMZ can occur without pushing by epiboly and convergence and extension of the NIMZ-AC; (2) involution and blastopore closure can occur without migration of the mesoderm on the blastocoel roof; (3) convergence and extension of the IMZ are sufficient to bring about IMZ involution and blastopore closure; (4) the function of bottle cells in initiating involution is retarded by presence of the NIMZ-AC; (5) the associated dorsal NIMZ and IMZ together form an axis that extends better and is perhaps stiffer than the IMZ alone; and (6) the dorsal axial and paraxial mesoderm form the "skeleton" around which the mechanics of the other parts of the embryo are organized. These findings are important for the analysis of gastrulation by cellular, genetic, and molecular approaches.


Cellular contribution of the different regions of the somatopleure to the developing limb.

Regionalization of the presumptive limb region was examined before and at the onset of limb development by means of a variety of transplantation experiments between quail and chick embryos in ovo. The results demonstrate a two-step process, the first of which is the designation of the region of the somatopleure that would become part of the limb, followed by specification of dorsal and ventral regions of the limb. The medial half of the somatic mesoderm is the region which gives rise to the limb with only a smaller cellular contribution from the lateral half of the somatic mesoderm. The cellular contribution of the medial region of the somatopleure appeared to determine the type of limb formed (i.e., wing or leg). The second process relates to changes in the ability of the somatic ectoderm to undergo extensive lateral displacement with development. Starting at stage 14, the medial and lateral somatic ectoderms maintain their position after transplantation, in contrast to earlier stage limb or flank ectoderms which undergo extensive lateral displacement with development. The positional determination of the dorsal and ventral properties of the medial and lateral ectoderms of the prospective limb region and their distal displacement during limb outgrowth may be important morphogenetic events in limb development.


Modifiable cardiac risk factors of obesity, inactivity, and stress: a community survey.

In this descriptive study the perceptions and practices of community residents concerning three risk factors associated with coronary heart disease (obesity, inactivity, and stress) and their prevalence were assessed. One thousand questionnaires were randomly mailed to residents of Thunder Bay, Ontario, Canada with a response rate of 48.1%. The results suggest that all three risk factors under study could be contributing to the significantly higher mortality rates in Thunder Bay due to coronary heart disease (when compared to provincial norms). Programs should be developed in the community under study to promote awareness of cardiac risk factors and strategies developed to reduce these risk factors.


The perceptions of stressors in coronary artery bypass graft patients.

A descriptive correlational study was conducted to compare the perceptions of 33 patients and their nurses of the stressors involved in the surgical procedure of Coronary Artery Bypass Graft (CABG). Five additional stressor items were added to the Stressor Scale developed by Carr (1982). Patients and nurses completed the questionnaires. Correlations and t tests were used to examine the relationship between the stressor scores. Overall, the nurses rated the stressors significantly higher than the patients. In the nursing group, nurses with more experience rated the stressors significantly lower than the nurses with less experience. These results were similar to the findings of Carr (1982). Implications for nursing theory, practice and research are discussed.


Cell interactions influence the pattern of biomineralization in the Ilyanassa obsoleta (Mollusca) embryo.

Ilyanassa obsoleta larvae have two calcium carbonate-containing organs, shell and statocyst, which are derived from five micromere cells (2a, 2c, 2d, 3c, 3d). "Internal shell," an abnormal, internal calcium carbonate mass, was previously observed when cells which normally induce shell and statocyst were removed. This study utilizes multiple-cell deletions to examine how these calcium carbonate-producing precursors control the pattern of biomineralization, whether it is in external shell, statocyst, or internal shell. It was demonstrated that internal shell was solely derived from any of these five cells. However, there was a quantitative difference in the frequency of internal shell production depending upon which cells, as well as how many, are deleted. In general, when external shell or statocyst production was diminished, as the result of removing several of the calcium carbonate-producing cells, internal shell was deposited instead. The presence of internal shell can best be explained as the result of altered interactions between these five cells after one or more have been deleted. Electron diffraction and transmission electron microscopy show that internal shell differs from normal shell in both structure and crystal morphology and it can also be produced by statocyst precursors. Thus, both the deletion and electron microscopy data support the interpretation that the development of internal shell is controlled by shell- and statocyst-producing cells when the cell communication between these cells is disrupted.


Stripe selection: an intrinsic property of some pattern-forming models with nonlinear dynamics.

In two-dimensional pattern formation, the genesis of stripped rather than spotted patterns may involve preexisting spatial asymmetries, such as unidirectional gradients or asymmetric shape of the pattern-forming domain. In the absence of such asymmetries, some kinds of nonlinear dynamics still lead to striped rather than spotted patterns. We have studied the latter effect both by extensive computer experiments on a range of nonlinear models and by mathematical analysis. We conclude that, when the dynamic equations are written in terms of departure from the unpatterned state, the presence of nonlinearities which are odd functions of these departures (e.g., cubic terms) together with absence of even nonlinearities (e.g., quadratic terms) ensures stripe formation. In computer experiments, we have studied the dynamics of two-morphogen reaction-diffusion models. The mathematical analysis presented in the Appendix shows that the same property exists in more generalized models for pattern formation in the primary visual cortex.


Neurofibromin, a predominantly neuronal GTPase activating protein in the adult, is ubiquitously expressed during development.

The onset of manifestations of the common, autosomal dominantly inherited disease type 1 neurofibromatosis (NF1) is usually in childhood. To begin to understand the pathogenesis of NF1, we analyzed the developmental pattern of expression of the protein product of the NF1 gene, neurofibromin, by Western blotting and immunohistochemistry using the rat as a model system. Neurofibromin is uniformly distributed throughout embryonic day 10 and 12 rat embryos. By embryonic day 16, neurofibromin immunoreactivity is enriched in neurons of the cortical plate, in peripheral ganglia, and in developing CNS and PNS fiber tracts, but remains detectable outside the nervous system. Expression decreases in nonneural tissues by postnatal day 6, and neurofibromin is greatly decreased (lung, adrenal cortex, skin) or absent (skeletal muscle, cartilage) in adult tissues except for brain, spinal cord, peripheral nerve, and adrenal medulla. Transient expression of neurofibromin during development in many tissues suggests the importance of this GTPase-activating protein in morphogenesis and organ growth. A separate role is proposed for neurofibromin in growing axons and in the mature nervous system.


[Development of an integrated software program to analyze statistically the results of complex investigations in space biology and medicine].

A concept of implementing the problem-oriented statistical software based on an application of expert systems is presented. The shortcomings of existing statistical software for personal computers and trends of their improvements are analyzed. Recommendations on designing of statistical expert systems can be used by the statisticians, mathematicians, programmers and users not being professionals in these fields. Integrated statistical software will make it possible to perform additional retrospective analysis and to check up the large arrays of previously gained data and to obtain supplementary scientific information and to find new regularities in the previous experimental findings.


[Pattern of spontaneous oculomotor activity in weightlessness and the readaptation period].

The results of one part Spontaneous Oculomotor Activity Study of Soviet-Bulgarian experiment "Labyrinth" conducted with the participation of 5 cosmonauts prior to and after 10- and 241-day flight are presented. Two cosmonauts have also been studied in-flight. The experiment was conducted with the use of automated data controlling and processing system "Zora" developed by the Bulgarian specialists on a personal computer basis. The horizontal and vertical electrooculograms were recorded in the subjects with eyes closed, open and open but shaded by opaque glasses with the gaze directed forward and in extreme left, right, up and down directions of the gaze. It is found that in the first 3 day of space flight especially when locking visual input, there is an increase in spontaneous activity of "swimming" and saccadic nature or spontaneous nystagmus mainly in the vertical direction. There have been sharp decreases in the rate of eye movement and increase of an amplitude when placing the eyes in the extreme positions (especially in up-down directions) accompanied by correcting saccades or adaptive nystagmus. The post- and inflight changes in the severity and pattern of spontaneous oculomotor responses were similar.


[Adaptation of young pilots to new conditions of professional activities (socio-psychological aspects)].

An adaptation of 146 young pilots to the flying career conditions was studied. The pilots filled in the questionnaire containing the points on: motivation, flight duty satisfaction, self-assessment of professional advancements and conditions under which it occurs and an attitude to flying personnel of the unit. The experimental findings can be used to improve the psychological relations of the young pilots during their adaptation to the new conditions of professional activity.


[Hemodynamic status of man in a graded orthostatic test].

Fourteen volunteers were exposed to a graded orthostatic test by a passive tilt from the horizontal position (0 degree) to an orthostatic posture (70 degrees) with intermediate angles of 10, 20, 30, 40, 50 and 60 degrees. Each position was maintained for 5 min. At the end of each step ECG, the first derivative of impedance cardiogram and auscultative blood pressure were registered. Stroke volume, cardiac output, Q-Z interval, thoracic impedance and diastole-systolic coefficient of impedance cardiogram (the parameter of pre-loading) were most markedly changed at the beginning of the test, when the tilt angle was less than 30 degrees. Its further increase was associated with moderate changes of the mentioned parameters. Diastolic arterial pressure began growing significantly from the tilt angle of 10 degrees and the cardiac rhythm became significantly more frequent starting from 30 degrees. Thus, the set of parameters registered becomes more informative if the test is performed in a graded manner since in this case it is possible to evaluate the order and magnitude of passive and active hemodynamic changes, and on the basis of their comparison to assess the adequacy of compensatory circulatory responses to orthostatic exposure.


[Maximal anaerobic capacity of man in a modified Wingate test].

We studied the possibility of using a 380B Siemens-Elema (Sweden) bicycle ergometer to determine the maximal anaerobic capacity of healthy subjects during a modified Wingate test. Exercise was performed under stable moment conditions, with calculation of braking resistance on the basis of the subjects lean body mass. The values of total work performed and maximal power may be used for comparative evaluation of physical work capacity in participants of training and rehabilitation programs.


[Informative value of rerespiration as a method for evaluating functional reserves of the cardiorespiratory system in modeling of some flight factors].

This article presents a study of the informative value of the recurrent breathing (rerespiration) method developed by the authors, for evaluating the functional status of the human body during recovery after exposure to noise and vibration, to piloting accelerations and head-down hypokinesis. It is shown that a 1.5-h exposure to noise and vibration increases tolerance to rerespiration, suggesting a stimulatory effect. A 5-h exposure in half of cases either does not change tolerance to the test or lowers it. Piloting head-to-seat accelerations of 7 to 9 G are associated with a lowering in tolerance to rerespiration, the functional status being recovered 1 d later. A similar exposure to 3-5 G in 83% of cases is not associated with an alteration of response to the test. After a 30-day head-down hypokinesis there is a lowering of tolerance to rerespiration, both in subjects with drug correction and in the control group. On day 10 of recovery in the control group return to the baseline rerespiration tolerance level was incomplete. Medically treated subjects demonstrated a complete recovery. Thus, rerespiration in the developed methodological version, is an informative loading test allowing to evaluate the dynamics of the functional status of the body. As a signal method it helps to clarify the individual duration of recovery after performing flight-related tasks, to substantiate the necessity of rehabilitation procedures and assess their effectiveness, as well as to detect persons with a stable decline of tolerance to rerespiration.


[Role of ultraviolet radiation and vitamin D metabolism in medical care during space flights].

Data on the development of ultraviolet (UV) deficiency in the cosmonauts are analyzed, possible ways to optimize UV radiation and alimentary stearin supply in order to prevent vitamin D deficiency and to correct the changes in phosphorous-calcium metabolism are discussed.


[Oxygenation of the frontal cerebral cortex in monkeys during a two-week space flight].

In monkeys (Macaca mulatta) during a two-week space flight and in a ground-based control experiment, with semi-closed platinum electrodes, partial oxygen pressure (pO2) was registered in the frontal cerebral cortex. It was shown that during the flight there is a marked increase of pO2 in the frontal cortex, reaching maximal values on days 5-8 (up to 203%) with a subsequent tendency to normalization by day 11. In the control experiment pO2 values in the cortical zone studied did not exceed the baseline level throughout the period of registration. Analysis of the curves of pO2 oscillations in the frequency range from 0.2 to 0.01 Hz in-flight revealed a shift of the oscillation power spectrum in the long-wave direction which was not seen in the control experiment and suggested a decreased rate of tissue metabolism. As a possible explanation for the noted effects of pO2 dynamics during weightlessness, a combination of a decreased metabolic rate in the brain segment under study with a disorder in the mechanism of regulation of local cerebral blood flow is suggested.


[Effects of hypokinesia on cardiac activity in rats with high and low spontaneous motor activity].

Hypokinesia effects on cardiac contractivity and vegetative regulation were studied in rats with high and low spontaneous motor activity. It was shown that hypokinesia promotes depression of the stroke volume and decrease of sympathetic nervous activity in rats with low motor activity. At the same time, in rats with high motor activity these parameters were not significantly affected by hypokinesia. The conclusion is made about a relationship between resistance to hypokinesia and spontaneous motor activity.


[Accumulation of calcium ions in myocardial sarcoplasmic reticulum of restrained rats exposed to the pulsed electromagnetic field].

Alteration in the velocity of Ca++ accumulation was investigated in myocardial homogenates from restrained rats which were chronically (1 and 2 months) exposed to the impulse electromagnetic field with frequency 1 Hz and magnetic induction 6-24 mT. The fall of the studied variable by 33% was observed in a month while restoration of the velocity of Ca++ transport was shown after two months of exposure. The results are discussed in terms of the organism adaptation to physico-chemical and physiological impacts.


[Significance of comparing the results of caloric and vestibulometric rotation tests].

The use of modified, less laborious, easier endured vestibulometric tests made it possible to facilitate diagnosing Menière's disease and neurinomas of the vestibular nerve. Comparison of nystagmometric findings recorded in the same patient during caloric and rotation tests using the diagnostic model of a bithermal test permitted some assumptions to be made in relation to the mechanisms for nystagmogenesis associated with pathologic states in ear labyrinth and vestibular portion craniocerebral (VIII) nerve. It is found that in the majority of Meniere's disease cases (61%) there is a decrease in an intensity of caloric nystagmus induced by Lasix dehydration effect. This phenomenon can be explained by a reduced difference in energy levels of two complexes of vestibular nuclei and, therefore, by an attenuated afferents to peripheral compartment of vestibular system. In neurinoma of aural nerve, the phenomenon of vestibular recruitment (20%) was noted. The diagnostic model of a bithermal test makes it possible to explain an effect of smoothing out the nystagmic responses induced by an intensive stimulus when used a sinusoidal stepwise test. The observed vestibular recruitment results from the severe disorder of one of the afferent inputs due to destruction of induced by tumor pressure.


[Age factors and flight duration in chronic diseases].

Health disqualification is the main reason for stopping flying activity. The age limitations of flying activity have been not established. Nevertheless, the development of diseases and motivated continuation of flying activity are severely age-dependent. The cases of chronic diseases have been reported throughout the all flying careers of 1,433 pilots and navigators and disqualifying for flying activity for the health condition upon routine medical screening. The flying activity duration after a disease occurrence was found to be of bimodal distribution. The first peak coincides with the moment of an initially diagnosed illness, the second-delayed peak in elder individuals approximates the first one. Thus, in the persons in whom a chronic disease was initially diagnosed when they were twenty years old, this diagnosis was confirmed in 18-20 years and in 2-4 years in the persons in whom a disease was diagnos ++ when they were 35 years old. Thus, there are two factors greatly affecting the disqualification--a disease and age. Both these factors are closely interrelated.


[Rationale for using helium-neon laser in medical rehabilitation of patients with atrophy].

High resolution measurements of helium-neon laser spectra have been conducted about 0.64 mm wavelength. The singlet oxygen photo-generation was demonstrated to be responsible for the bioeffects of this spectral interval. Helium-neon radiation with 0.633 mm wavelength was used for medical rehabilitation of 15 patients with muscular atrophy as a result of wearing immobilizing plaster arm-bends on lower extremity fractures. 50-day hypokinesia of a damaged extremity induced differences in functional indices between damaged and healthy extremities in the same patient. This difference was diminishing more rapidly after laser therapy than in the control group though all patients had adequate rehabilitative treatment. Due to the likelihood of hypokinesia- and weightlessness-induced atrophies, helium-neon laser may be recommended for atrophy prophylaxis and treatment in both cases.


The CARES: a generic measure of health-related quality of life for patients with cancer.

In spite of the prevalence of neoplastic disorders as a cause of chronic illness, very few of the currently available generic measures of health-related quality of life or health status have been utilized with cancer patients. In this paper we reviewed our studies with the Cancer Rehabilitation Evaluation System (CARES), a cancer-specific measure of rehabilitation needs and quality of life. We present data to demonstrate that the CARES is a generic measure of health-related quality of life, suitable for use in the many different diseases called cancer. The data that form the basis for this paper were collected during a decade of research on the CARES. This paper represents the first discussion of the performance of the CARES across separate cancer sites and phases of the disease. In addition, we demonstrated that the CARES is responsive to changes in health-related quality of life over time. Finally, we will discuss the application of the CARES in clinical and research settings.


Is there a role for preference assessments in research on quality of life in oncology?

The development of ways to evaluate interventions that may have an impact on quality of life is a rapidly-developing area of research in clinical oncology, especially within the context of randomized controlled trials. We propose a role for assessments of preferences in such evaluations, including preference studies designed to assess attitudes toward the clinical acceptability of interventions, and preference trials designed to assess choice behaviour in relation to interventions. We suggest that such preference assessments represent a specific case of a more general issue: the need to develop an 'ethics of evidence', that is, standards for the creation, assessment and communication of evidence. We then outline a framework within which an 'ethics of evidence' might be developed, and suggest that the framework also may provide a useful model for the processes involved in the transfer of research results into clinical practice. As an illustration, we consider the problem of decision making in circumstances where the choice of therapy depends primarily on the patient's own preferences, as, for example, in the choice of mastectomy or breast-conserving treatment in early-stage breast cancer. The long-term goal is to develop criteria which might be used to foster shared rational decision making in such circumstances.


Assessing quality of life in head and neck cancer.

Comprehensive assessment of the impact of head and neck cancer goes beyond traditional biomedical outcomes to include quality of life. This paper examines the methods used in reported studies on quality of life in head and neck cancer patients with respect to definitions, approaches to measurement, and types of findings. Analysis reveals little consensus in defining or assessing quality of life, although deficits in patient well-being were well-documented. A number of current efforts to develop and validate quality of life instruments specific to cancers of the head and neck have been reported. Future research should be sensitive to the issue of defining quality of life; a definition which includes both patient perceptions and objective functioning in a number of areas is advisable. Investigators should build on recent and ongoing efforts at instrument development and make efforts to incorporate quality of life assessment in clinical trials of cancer treatments. The distinctive aspects of head and neck cancer illustrate the importance of considering characteristics of the patient population in quality of life research and also the way that quality of life data can contribute to the multidisciplinary team of caregivers.


Patient acceptance and differential perceptions of quality of life measures in a French oncology setting.

A three-part study evaluated French cancer patients' acceptance of self-rated quality of life measures, the predictive value of these measures, and the agreement between patient and health provider ratings of patient quality of life. In part one, 93% of 137 patients indicated a willingness to complete the Qualite de la Vie-Questionnaire (QOL-Q) and Analogues Lineaires pour la Mesure de la Qualite de vie (LA), and 63.6% indicated a willingness to be interviewed by a psychologist. Willingness to complete the scales was related to hospitalization status and treatment modality. In parts two and three, 100 patients were asked to complete the QOL-Q, LA, the Karnofsky Index (KI) and a side-effects checklist, and to undergo a psychological interview. Following interview, a psychologist rated the patients using the QOL-Q, and classified patient level of emotional distress. Oncologists rated the patient using the KI and the side-effects checklist. The results indicate that the patients' ratings of their quality of life were higher than the psychologist's ratings, and that the QOL-Q has predictive value in identifying severe emotional distress. Low level of patient and physician agreement on the KI was partially explained by patient age and cancer site. A moderate level of agreement was found between patient and physician perceptions of side effects. The study suggests that the QOL-Q, not the LA, may be useful as a screening tool to identify patients with impaired quality of life, and that self-rated measures should be included in quality of life assessments.


Reliability and validity of the Katz Adjustment Scales in an epilepsy sample.

Development of quality of life measures de novo is time-consuming and expensive, and a number of instruments are already available for general use. Reevaluation and refinement of quality of life tools are needed to improve the existing pool of measures. In this study, data from a sample of 328 epilepsy patients were used to revise a measure of social adjustment and emotional status developed in the 1960s, the 127-item form R1 of the Katz Adjustment Scales (KAS-R1). Using a comprehensive item analysis procedure, we increased the number of items used in scoring the KAS-R1 from 76 to 113 and substantially improved the reliability of scales in both the original sample of 328 epilepsy patients and in a second administration to a 'cross-validation' sample of 193 epilepsy patients. Support for the validity of the revised KAS-R1 scoring system was obtained in a known groups analysis of patients who had previously undergone surgery for epilepsy: mean scores were significantly higher (p < 0.05) on 11 out of 14 scales in the revised KAS-R1 for patients who were completely seizure-free compared to patients who were having seizures with loss of consciousness, after adjusting for age, gender, and anticonvulsant medication use.


Responsiveness to change: an aspect of validity, not a separate dimension.

Assessment of health-related quality of life is accelerating in naturalistic observational studies, clinical trials, and clinical practice. Some researchers have argued that the ability of a quality of life instrument to detect clinically important changes over time, "responsiveness," is a distinct psychometric property from the measure's reliability and validity. We discuss the important implications of this argument and counter that responsiveness is actually one indication of a measure's validity.


A short multilingual quality of life questionnaire--practicability, reliability and interlingual homogeneity.

International cooperative clinical trials require a multilingual quality of life questionnaire. The 'International Society for Chemo- and Immunotherapy' therefore designed a study to develop and test a 'health accentuated' quality of life questionnaire in the eight languages spoken in this society. The objective was to examine practicability, reliability and interlingual homogeneity. Versions of the questionnaire in Czech, German, Hungarian, Italian, Kroatian, Polish, Romanian and Slovakian have been prepared. The results are based on data of 1,104 adult patients. They demonstrate that the developed questionnaire is practicable. Patients need 10-15 min to answer it, usually without assistance. The proportion of missing values is for all but two questions less than 3%. The questionnaire also found high acceptance reflected by only 2.7% refusals. It is reasonably reliable in each language. Interlingual homogeneity could be shown by demonstration of strong structural similarities between the different versions using multidimensional scaling, factor analysis and comparison of mean profiles.


Psychosocial, neuropsychological and neurological status in a sample of heart transplant recipients.

The sample for this psychosocial follow-up study was formed using the 71 surviving patients from a total of 100 who underwent heart transplantation up until March 1990 at Kiel University Hospital. Forty of these could be investigated comprehensively. The neurological investigation comprised EEG and auditory and visually evoked potentials. The psychological investigation started with several neuropsychological tests; additionally all patients were interviewed and they completed questionnaires concerning anxiety, depression, ways of coping, personality characteristics and life satisfaction. Within the entire sample, there were few indications for specific psychological impairment. The psychosocial status of the patients was not significantly related to the time which had elapsed since they underwent transplantation. There were few associations between neuropsychological and psychosocial data, and several indicators of early postoperative complications which are described in the literature (e.g., length of stay in the intensive care unit). Clinically relevant subgroups (formed on the basis of neuropsychological test results, anxiety and depression) did not differ in terms of these indicators. Nevertheless, a considerable proportion of the patients (25%) were characterized by having a higher level of affective and neuropsychological impairment. Although based upon retrospective investigations, the results of this follow-up generally correlate with those gained from Anglo-American countries. In interpreting the results one needs to consider the possible influence of premorbid personality characteristics as well as denial which may be typical for patients undergoing heart transplantation.


Assessment of validity and clinical application of an Italian version of the Rotterdam Symptom Checklist.

An Italian version of the Rotterdam Symptom Checklist (RSCL) has been validated in a sample of 147 healthy women and 61 breast cancer outpatients. The structure of the RSCL has been analysed by factor analysis reproducing results of the English version, and the psychological and physical subscales showed a good internal reliability (Chronbach's alpha 0.91; 0.87). The State-Trait Anxiety Inventory questionnaire and the RSCL were compared in the healthy women sample. A Pearson correlation coefficient (PCC) of 0.74 (p < 0.01) was found between the trait anxiety and RSCL psychological scores. Psychological (mean: 17.5; SD: +/- 5.4) and physical (mean: 37.8; SD: +/- 8.6) subscale scores were correlated in healthy women (PCC: 0.68; p < 0.01) and related to a general wellbeing item (PCC: 0.44; p < 0.01). The breast cancer outpatients showed scores for psychological and physical subscales similar to those in the healthy women sample. The Italian version of the RSCL seems comparable to the English version and could be useful in clinical research. Its use in longitudinal studies should be tested.


Severity of disease and quality of life: a comparison in patients with cancer and benign disease.

The aim of our study was to compare the quality of life in patients with malignant and benign diseases. Eighty-nine females and 110 males (mean age 59 years) from the surgical unit at the University Hospital of Linköping were examined. Ninety-five subjects suffering from a gastrointestinal malignancy were compared with 104 non-malignant patients, of which 57 had benign gastrointestinal disorders, 24 endocrine and 23 vascular diseases. Quality of life was defined as perceived well-being and life satisfaction, globally as well as within key domains. Two separate types of self-ratings, a life domain (20 items) and a well-being rating (eleven subscales, each scale with ten items), were performed in the medical ward. The life domain rating covered the subject's own appraisal of his health and psychological circumstances; social network; activities and habits; of a structural domain ('ability to lead a meaningful life') and of his global life satisfaction. The well-being rating focused on emotional experiences (basic mood, security, indolence, loneliness, inferiority, and guilt together with future-orientation) and perceived behaviour (endurance, tension, sociability and attitudes of the environment). The patients who had a gastrointestinal malignancy scored consistently 'lower' than patients with benign diseases. After adjusting for age concerns, most of the significant inter-group differences remained. After adjusting for nutritional state influences (e.g., recent weight loss), differences in global life satisfaction, future-orientation and perceived attitude from the environment were still apparent. This study is contrary to several other studies undertaken in search of expected group differences but none were found.(ABSTRACT TRUNCATED AT 250 WORDS)


Issues of concern in the standardization and harmonization of drug trials in Europe: health-related quality of life, ESCT meeting, Strasbourg, 23-24 May 1991.

Overviews of the development of the concept of quality of life from a medical viewpoint, and the need for new models and methods to assess health-related quality of life were presented, along with WHO, FDA and European regulatory viewpoints and a discussion on the measurement and valuation of health by social judgement, at the health-related quality of life session at an ESCT meeting on issues of concern in the standardization and harmonization of drug trials in Europe. It was concluded that, for the present, all three types of measurements of quality of life--individual, disease-specific, and generic scales--should be used in research to look for convergent versus divergent validity.


An overview of generic health-related quality of life measures for HIV research.

This paper provides a selective review of instruments currently being employed to evaluate generic health-related quality of life in studies of persons with human immunodeficiency virus (HIV). Instruments examined include the Quality of Well-Being Scale, the HIV Overview of Problems/Evaluation System, the COOP Charts, and six Medical Outcomes Study-based measures: the SF-20, SF-30, AIDS-HAQ, SF-36, SF-38 and SF-56. Relative strengths and weaknesses of the measures are discussed.


The short-term effect of patient health status assessment in a health maintenance organization.

This study was designed to test the short-term effects of health assessment on the process of care and patient satisfaction. The 29 Chart physicians used the Dartmouth COOP Charts to measure their adult patients' health status during a single clinical encounter; the 27 control clinicians used no measure of health status. We compared the change between baseline and post-intervention information for a sample of all study clinicians' patients. Most of the patients were female (67%), well educated (70% had at least a college education) and young (approximately 90% were aged 59 years or younger). We found that the ordering of tests and procedures for women was increased by exposure to the COOP Charts (52% vs. 35%; p < 0.01); the effect in men was not as significant (37% vs. 23%: p = 0.06). Although women reported no change in satisfaction with care, men claimed that the clinician helped in the management of pain (p = 0.02). We conclude that the use of health status measures during a single clinical encounter in an HMO changes clinician test ordering behaviour and may improve the help male patients receive for pain conditions. The long-term impact of these management changes is not known.


Uncertainty, appraisal and quality of life.

This study evaluates the influence of different factors in the adaptation process activated by uncertainty in illness on health-related quality of life. The sample included 100 women (mean age = 52.1 years) receiving treatment for newly diagnosed (M = 5.1 months) gynaecological cancer (38 cervical, 26 ovarian, 24 endometrial, 7 uterine, 4 vulvar, and 1 vaginal). Stepwise regression analyses identified mood states, ambiguity about illness-wellness state, danger-focused appraisal and mastery as key predictors of four health-related quality of life scores. The variance accounted for by those variables is reflected in cumulative multiple R2 of 0.56 for total quality of life score, 0.57 for psychosocial well-being, 0.235 for physical well-being and 0.25 for disease/symptom distress. These variances do not reflect the contribution of age, time since diagnosis, metastasis and stage of cancer which were forced to enter the regression equation first. The latter set of variables accounted for a smaller portion of the variance in health-related quality of life (R2 = 0.03-0.195). Coping strategy did not predict health-related quality of life. These findings provide beginning support for conceptualizing health-related quality of life as the outcome of an adaptation process explained by the uncertainty in illness theory. However, uncertainty in illness theory may not be sufficient to predict quality of life outcomes. Future research should consider the addition of discrepancy theory to guide the selection or development of a health-related quality of life measure, to account for the perceived discrepancy between actual experience and expected well-being.


Quality of life during rehabilitation after coronary artery bypass surgery.

The effect of a three-phase comprehensive rehabilitation programme on the quality of life during the first postoperative year after coronary artery bypass surgery was studied in 205 male patients randomly allocated into a rehabilitation (R) and a hospital-based treatment (H) group. The rehabilitation programme included physical exercise, relaxation training, psychological group sessions, dietary advice and discussions about postoperative treatment of coronary disease. There was no difference between R and H groups in the frequency of postoperative complaints, number of hospital admissions and satisfaction of sexual life. An almost significantly greater number of subjects in R group than in H group perceived their health as good 12 months after surgery. The Beck Depression Index score decreased significantly in R group but not in H group during follow-up. A greater increase in hobby activities was observed in R group than in H group. More subjects in R group than in H group considered rehabilitation important for recovery, whereas more patients in H group considered support by the spouse and family, the subjective mental strength and a secure income as important.


Clinical outcome and emotional-behavioural status after isolated coronary surgery.

In order to evaluate clinical and psychosocial results of isolated coronary artery by-pass graft (CABG) we studied 626 consecutive patients, mean age 61 +/- 8 years (86% men), in a follow-up (median: 58 months) with a complete questionnaire about cardiosurgical problems (post-operative vital status, angina relapse, infarction, heart failure, PTCA, redo, PM) and psychosocial variables (mood, irritableness, job satisfaction, hobby satisfaction, family relations, sexual activity, general well-being and work status). Global evaluation improvement of psychosocial variables was found in 71% of patients without cardiac events (group A) and 11% of patients with cardiac events (group B); worsening was found in 2% of group A and 1% of group B; no referred variations in 13% and 2% respectively (p < or = 0.05. Interests (in work, hobbies and sexual activities) demonstrate an improvement in 20% (group A) and 2% (group B); worsening in 12% (group A) and 4% (group B); no variations in 51% (group A) and 11% (group B) (p < or = 0.005). Patients reported a well-being evaluation improvement about 66% in the group returning to work without restriction, 13% in those with limitation, 6% no further working; worse or unchanged well-being evaluation was found in 9% of patients returning to work without restriction, 3% with limitation, 3% no further working (p < or = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Methods for assessing quality of life in the cardiac arrhythmia suppression trial (CAST).

The CAST was a randomized, double-blind placebo-controlled multicentre trial of antiarrhythmic medications designed to suppress ventricular arrhythmias in patients after an acute myocardial infarction (MI). A collection of 21 items derived from established scales was used to assess aspects of quality of life in CAST. The questions focused on symptoms, mental health, physical functioning, social functioning, life satisfaction, and life expectancy. Additional aspects included exposure to major stressful life events, and perceived social support and social integration. Work status was also recorded. Using the baseline values of 1465 (98%) out of 1498 patients enrolled in the CAST main study between 15 June 1987 and 19 April 1989, the reliability and validity of the scales used in CAST were computed. High internal consistency reliability (> or = 0.70) was found for Symptoms, Mental Health, and Physical Functioning. The discriminative validity, in particular for Symptoms, Mental Health, Physical and Social Functioning, showed that patients with heart failure and previous MI, as well as those suffering from angina and dyspnea, had a worse quality of life than those patients who were not experiencing these symptoms. It was concluded that the scales selected to form the CAST quality of life questionnaire were both reliable and clinically valid for this patient population and therefore could be used to detect disease progression and treatment effects.


The Quality of Life Committee of the Clinical Trials Group of the National Cancer Institute of Canada: organization and functions.

The Quality of Life Committee of the National Cancer Institute of Canada Clinical Trials Group has successfully advocated the adoption of quality of life outcomes in Canadian clinical trials in patients with cancer. It has developed a policy promoting quality of life assessment in phase III trials and developed writing guidelines to assist clinical investigators when developing protocols for proposed studies. As a result, all phase III clinical trials instituted since the policy was developed have quality of life assessment as a part of their objectives. It has assisted the Clinical Trials Group office personnel in the development of procedures for the conduct of quality of life assessments in clinical trials with the result that the completion rate of self-report questionnaires is very high. The Committee maintains contact with other clinical trials groups and organizations having an interest in measuring quality of life in cancer.


Statistical analysis of longitudinal quality of life data with missing measurements.

The statistical analysis of longitudinal quality of life data in the presence of missing data is discussed. In cancer trials missing data are generated due to the fact that patients die, drop out, or are censored. These missing data are problematic in the monitoring of the quality of life during the trial. However, by means of assuming that the cause of the missing data lies in the observed history of the patients and not in their unobserved future, the missing data are ignorable. Consequently, all available data can be used to estimate quality of life change patterns with time. The computations that are required are illustrated with real quality of life data and three commonly used computer packages for statistical analysis.


Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.


Towards the molecular localisation of the AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome.

We have used a series of 30 DNA probes previously mapped to the long arm of the human Y chromosome, to screen a panel of 21 patients with structural abnormalities in Yq, by genomic blot hybridisation. The results have allowed us to construct a detailed map of interval 6 of the Y chromosome, in which 28 of the probes could be assigned to 14 sub-intervals within interval 6. Some probes detect two or more loci within this region, each of which has been localised. The same set of probes has been used to screen a panel of 19 chromosomally normal azoospermic men, two of whom have been found to carry microdeletions within this region. With the completion of this map we have been able accurately to localise these microdeletions within interval 6 and show that they do not overlap. We believe these microdeletions may disrupt the azoospermia factor (AZF) involved in spermatogenesis, and which is known to lie in this region. These results are an important step towards the localisation of the AZF locus.


Beginning or end? Telomere structure, genetics and biology.

The word telomere derives from the Greek word telos meaning 'end', roughly translating as 'the thing at the end' when the end is that of a chromosome. Telomeres belie their apparent simplicity of structure by being involved in a wide range of diverse biological phenomena. Much of our understanding of telomere behaviour comes from studies in lower eukaryotes such as ciliates and yeast, the subject of many recent reviews. Here we concentrate on the mammalian telomere, recent progress in its study, and how recent evidence for an involvement of telomeres in the regulation of gene expression and DNA replication in yeast points to new aspects of mammalian telomere function yet to be explored.


Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice.

Duchenne and the less severe Becker form of muscular dystrophy (DMD,BMD) result from genetic deficiency in the level and/or activity of the protein dystrophin. The recent availability of cDNA based minigenes encoding recombinant dystrophin polypeptides has raised the possibility of somatic gene transfer as a therapeutic approach to treat dystrophin deficiency. In this respect, the mdx mouse provides a useful model of DMD exhibiting features characteristic of both the early myopathic and later fibrotic phases of the human disease. Using a mutated human cDNA, compatible in size with virus-based somatic gene transfer vectors, the pathophysiological consequences of restoring dystrophin expression have been examined in transgenic mdx mice. Transgene expression was correlated with a marked reduction of the skeletal myofibre necrosis and regeneration which is a major feature of the dystrophin-deficient phenotype in young mdx mice. The cDNA construct which is based on a very mild BMD phenotype thus encodes a highly functional dystrophin molecule whose reduced size renders it an attractive candidate for development as a therapeutic gene transfer reagent.


A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa.

Recently it has been demonstrated that some families with autosomal dominant retinitis pigmentosa (adRP) have mutations in the rhodopsin gene while others do not. Previously we have identified six such mutations in seven adRP families in this laboratory, one of which was previously described in US patients. We now present a completed screen of the rhodopsin gene in a panel of 39 adRP families, by a rapid screening technique which will be of use for routine diagnosis. Nine different mutations were ultimately found, in a total of twelve of the 39 families. These include the six previously identified mutations, in codons 68-71, 190, 211, 255, 296 and 347, two new ones in codons 53 and 106, and another mutation first identified in a single US patient, in codon 58. Thus approximately 30% of adRP families have 'Rhodopsin RP' while the remainder probably have a defect elsewhere in the genome. Of those families in which rhodopsin mutations have been found, four have been classified D type, three as sectorial RP and the remainder are of uncertain classification. All families excluded from chromosome 3q by linkage have been classified R type. These data suggest a correlation between clinical sub-classification and the underlying rhodopsin/non-rhodopsin heterogeneity.


A gene subject to genomic imprinting and responsible for hereditary paragangliomas maps to chromosome 11q23-qter.

Paragangliomas of the head and neck are slow growing tumors which rarely show malignant progression. Familial transmission has been described consistent with an autosomal dominant mode of inheritance. Clinical manifestations of hereditary paragangliomas are determined by the sex of the transmitting parent. All affected individuals have inherited the disease gene from their father, expression of the phenotype is not observed in the offspring of an affected female until subsequent transmittance of the gene through a male carrier. This finding strongly suggests that genomic imprinting is involved. We report the results of a linkage study on a large Dutch pedigree with hereditary paragangliomas. Highly significant evidence for genetic linkage to chromosome 11q23-qter with the anonymous DNA marker D11S147 was detected with a peak lod score of 6.0 at a recombination fraction theta = 0.0. Likelihood calculations yielded an odds ratio of 2.7 x 10(6) in favor of genomic imprinting versus the absence of genomic imprinting.


Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells.

The 14kb dystrophin transcript from the Duchenne muscular dystrophy (DMD) locus, which encodes a 427kDa protein, is differentially spliced at the amino terminal end giving rise to alternative transcripts expressed in muscle and brain. Here we present evidence for a 4.8kb transcript from the DMD locus which is ubiquitously expressed but is particularly abundant in Schwannoma cells where dystrophin could not be detected. The hybridisation of Western blots with dystrophin antibodies also identifies a protein of approximately 80kDa of variable abundance in different human and mdx tissues. Immunocytochemistry studies confirm the expression of this protein in nerve cells, a tissue in which full length dystrophin is not detected. Sequencing of the 5' end of a clone isolated from a rat Schwannoma cDNA library, shows that the 4.8kb transcript shares exons with the carboxy terminal end of dystrophin but the 5' untranslated region is not contained within the dystrophin transcript. We propose that the 4.8kb gene product be called apodystrophin-1 as its expression is distinct from the dystrophin 14kb mRNA but it is transcribed from the same locus.


Characterisation of a new rare fragile site easily confused with the fragile X.

A new fragile site (FRAXE) in Xq28 is described. It appears to be a typical folate sensitive fragile site. The fragile site is not associated with mental retardation, it does not give abnormal results when subjected to Southern analysis with probe pfxa3 which detects the unstable DNA sequence characteristic of fragile X syndrome. In situ hybridization mapping locates the fragile site between 150 kb and 600 kb distal to FRAXA. The distinction between the two fragile sites is important clinically since cytogenetic detection of FRAXE, without molecular analysis, could result in misdiagnosis of fragile X syndrome.


A male with two contiguous inactivation centers on a single X chromosome: study of X inactivation and XIST expression.

This report investigates the case of a patient with a duplication of the long arm of the X chromosome containing the putative X inactivation center (XIC) and the recently isolated XIST (X inactive-specific transcript) gene which is thought to play a role in the X inactivation process. Based on replication studies, a two-fold higher than normal phosphoglycerate kinase activity and absence of XIST expression, we conclude that the duplicated region is not subject to X-inactivation, despite the presence of two XICs. These results are discussed with respect to different models of X inactivation.


Isolation and characterization of 19 dinucleotide repeat polymorphisms on chromosome 3p.

We have screened cosmids on chromosome 3p for (dC-dA)n.(dG-dT)n dinucleotide-repeat sequences. Eighty-nine of 155 cosmids (58%) contained (dC-dA)n.(dG-dT(n repeats as determined by colony hybridization with a (dG-dT)10 oligonucleotide probe; 29 of these were subcloned and the sequences flanking the dinucleotide repeats were determined. Nineteen of the 24 loci examined for polymorphisms by PCR were found to be polymorphic with heterozygosities ranging from 3% to 86%. These dinucleotide repeat polymorphisms will be useful markers for high-resolution mapping of genes that have been localized to 3p, including tumour suppressor genes associated with several types of cancer and genes responsible for various hereditary disorders, such as von Hippel-Lindau disease.


An autosomal homologue of the choroideremia gene colocalizes with the Usher syndrome type II locus on the distal part of chromosome 1q.

Employing the mouse homologue of the human choroideremia cDNA as a probe, we have identified a homologous human gene. The consensus cDNA of this gene, designated human choroideremia-like (hCHML) gene, encompasses an open reading frame of 1968 base pairs. The deduced polypeptide of hCHML displays several regions of homology to smg p25A GDI, a bovine protein known to regulate the GDP/GTP exchange of the GTP-binding protein smg p25A. hCHML is located at 1q31-qter, a chromosomal region which, by means of linkage analysis, was previously shown to carry a gene locus for Usher syndrome type II. The colocalization of hCHML and Usher syndrome type II, as well as the clinical similarities between choroideremia and Usher syndrome type II, make hCHML a candidate gene for this disorder.


The Norrie disease gene maps to a 150 kb region on chromosome Xp11.3.

Norrie disease is a human X-linked recessive disorder of unknown etiology characterized by congenital blindness, sensory neural deafness and mental retardation. This disease gene was previously linked to the DXS7 (L1.28) locus and the MAO genes in band Xp11.3. We report here fine physical mapping of the obligate region containing the Norrie disease gene (NDP) defined by a recombination and by the smallest submicroscopic chromosomal deletion associated with Norrie disease identified to date. Analysis, using in addition two overlapping YAC clones from this region, allowed orientation of the MAOA and MAOB genes in a 5'-3'-3'-5' configuration. A recombination event between a (GT)n polymorphism in intron 2 of the MAOB gene and the NDP locus, in a family previously reported to have a recombination between DXS7 and NDP, delineates a flanking marker telomeric to this disease gene. An anonymous DNA probe, dc12, present in one of the YACs and in a patient with a submicroscopic deletion which includes MAOA and MAOB but not L1.28, serves as a flanking marker centromeric to the disease gene. An Alu-PCR fragment from the right arm of the MAO YAC (YMAO.AluR) is not deleted in this patient and also delineates the centromeric extent of the obligate disease region. The apparent order of these loci is telomere ... DXS7-MAOA-MAOB-NDP-dc12-YMAO.AluR ... centromere. Together these data define the obligate region containing the NDP gene to a chromosomal segment less than 150 kb.


Isolation and characterization of a novel gene with differential expression in benign and malignant human breast tumours.

We report the identification of a novel cDNA representing an mRNA showing significantly higher levels of expression in benign breast lesions than in carcinomas. This cDNA was identified by differential screening of a cDNA library generated from a breast carcinoma, and shows consistently higher expression in fibroadenomas than in carcinomas. The expression in both benign and malignant tissues is highest in epithelial cells as determined by in situ hybridization to tissue sections. The nucleotide sequence of the full-length cDNA has been determined, and the deduced protein is highly basic with no signal or transmembrane sequence, but two potential nuclear localization signals. Neither the DNA nor the protein sequence show any significant homology to sequences in current databases. The cDNA hybridizes to multiple sequences within both human and other mammalian genomes, but to single genomic sequences in Drosophila, Physarum and Schizosaccharomyces pombe. This cDNA therefore represents a highly conserved gene sequence. We have identified only one major transcript in human cells, and it seems likely that there are several pseudogenes within the human genome.


The gene encoding the palmitoylated erythrocyte membrane protein, p55, originates at the CpG island 3' to the factor VIII gene.

A CpG island 30 kb 3' to the human factor VIII gene in Xq28 is associated with a 2-kb transcript. This gene encodes a previously described palmitoylated membrane protein, p55, containing a src homology motif, SH3. Although originally described in reticulocytes, the transcript is expressed in a wide variety of human tissues. The gene is also present in the mouse and expressed in all mouse tissues examined. No known factor VIII gene deletions extend into the p55 gene. Since the function of the p55 protein is not known, the p55 gene is formally a candidate for any of the 19 or more disease genes that have not been isolated but are closely linked genetically to the factor VIII gene.


The organization of the intron-containing human S6 ribosomal protein (rpS6) gene and determination of its location at chromosome 9p21.

The intron-containing gene encoding human ribosomal protein S6 (rpS6), the major phosphoprotein in the mammalian ribosome, has been cloned. Using a PCR based cloning strategy we have isolated the rpS6 intron-containing gene in the presence of its many processed pseudogenes and determined the DNA sequence of the entire gene and its upstream and downstream flanking regions. The human rpS6 gene is 3979 bp in length and comprises six exons. Despite lacking a consensus TATA box, primer extension analysis indicates that the start of transcription is located at a single C residue within an 11 bp oligopyrimidine tract. The first exon, which contains the ATG start codon, is 48 bp in length. The DNA sequence in the 5' region of the gene has features of a CpG-rich island. Using fluorescence in situ hybridization (FISH) analysis the position of the rpS6 gene has been sublocalized to human chromosome 9p21. The similarities and differences between rpS6 and other previously characterized ribosomal protein genes are discussed.


Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)


A YAC contig in Xp21 containing the adrenal hypoplasia congenita and glycerol kinase deficiency genes.

The gene loci for adrenal hypoplasia congenita (AHC) and glycerol kinase deficiency (GK) map in Xp21 distal to Duchenne muscular dystrophy (DMD), and proximal to DXS28 (C7), by analysis of patient deletions. We have constructed a yeast artificial chromosome (YAC) contig encompassing a 1.2 Mb region extending distally from DMD, and containing DXS708 (JC-1), the distal junction clone of a patient with GK and DMD. A pulsed-field gel electrophoresis map of the YAC contig identified 3 potential CpG islands. Whole YAC hybridization identified cosmids both for construction of cosmid contigs, and isolation of single copy probes. Thirteen new single copy probes and DXS28 and DXS708 were hybridized on a panel of patients; the deletion mapping indicates that the YAC contig contains both GK and at least part of AHC, and together with the physical map defines a GK critical region of 50-250 kb. In one AHC patient with a cytogenetically detectable deletion we used the new probes to characterize a complex double deletion. Non-overlapping deletions observed in other unrelated AHC patients indicate that the AHC gene is large, extending over at least 200-500 kb. This mapping provides the basis for the identification of the AHC and GK genes.


High-resolution in situ hybridization using DNA halo preparations.

To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 microns, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5-10 cells only. The resolution range of the technique is at least 10-200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.


Multiple colors by fluorescence in situ hybridization using ratio-labelled DNA probes create a molecular karyotype.

Fluorescence in situ hybridization (FISH) is now widely used for the localization of genomic DNA fragments, and the identification of chromosomes by painting. We now show that half of the chromosomal complement can be painted in twelve different colors by using human chromosome specific libraries carrying three distinct labels mixed in multiple ratios. The photographs are in 'real' color rather than 'colorized'. The painting technique described here can be used for the identification of small or complex chromosomal rearrangements and marker chromosomes in humans or in any other species for which well defined chromosome specific libraries exist in a laboratory equipped with a conventional fluorescence microscope. The versatility of this novel cytogenetic technology may well constitute an advancement comparable to the introduction of chromosome banding and high resolution analysis of chromosomes in prometaphase.


A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.


Construction of a physical map on mouse and human chromosome 1: comparison of 13 Mb of mouse and 11 Mb of human DNA.

Long range restriction site maps of 13 Mb of mouse chromosome 1 and 11 Mb of human chromosome 1 were constructed using a framework provided by a detailed mouse genetic map. Where an unambiguous gene order could be determined in both species (14 genes), the human and mouse orders were identical. In addition, the distances between markers in the mouse and human were similar except for one region of the conserved linkage group where we could detect a larger distance in the mouse compared to the human. These data support the use of comparative mapping in physical map construction and further suggest the value of using mouse genetics to help define human disease loci.


Variable breakpoints in Burkitt lymphoma cells with chromosomal t(8;14) translocation separate c-myc and the IgH locus up to several hundred kb.

In about 80% of Burkitt's lymphoma cases, the tumour cell harbours a reciprocal chromosomal translocation which invariably transposes the coding exons 2 and 3 of c-myc from chromosome 8 to the immunoglobulin heavy chain locus on chromosome 14. Those t(8;14) translocations which disrupt chromosome 8 within or close to the c-myc gene are well documented. In this study we have focussed on t(8;14) translocations with the chromosomal breakpoint far upstream of c-myc. We analyzed the breakpoint position in 44 BL cell lines with t(8;14) translocations of different geographical origin and identified 9 cell lines with the breakpoint more than 14 kb upstream of c-myc. In these cell lines the positions of the translocation junctions on the derivative chromosomes 8q- and 14q+ were mapped by pulsed field gel electrophoresis and multicolour fluorescence in situ hybridization. The breakpoints occur at distances between 55 and more than 340 kb upstream of c-myc with no preferential site on chromosome 8. On chromosome 14, however, the translocation breakpoints are clustered in a narrow region 5' of the intron enhancer of the immunoglobulin heavy chain gene. In 7 of 9 cases, the enhancer is fused to the c-myc bearing sequences of chromosome 8. In two cases, the translocation has occurred in switch mu and downstream of C mu, respectively. The impact of these results with respect to the hypothesis, that cis-regulatory sequences from the immunoglobulin heavy chain locus can deregulate c-myc expression in a manner sufficient for tumour formation, is discussed.


Identification of a highly polymorphic marker within intron 7 of the ALAS2 gene and suggestion of at least two loci for X-linked sideroblastic anemia.

We have identified a compound dinucleotide repeat within intron 7 of the human erythroid 5-aminolevulinate synthase (ALAS2) gene with a minimum of 9 alleles and heterozygosity of 78%. ALAS2 was placed on the multipoint linkage map of the X chromosome in the pericentromeric region with the locus order: pter-(DXS255, TFE3, DXS146)-(DXS14, ALAS2, DXZ1)-AR-(DXS153, DXS159)-qter. No recombination was observed between ALAS2 and the centromere marker DXZ1. As ALAS2 has recently been shown to be the defective locus in X-linked pyridoxine-responsive sideroblastic anemia (PRSA), the ALAS2 marker has allowed placement of the gene for PRSA into the multipoint linkage map of the X chromosome. With the previous exclusion of close linkage between DXS14 and sideroblastic anemia with ataxia, our data show that there are at least two loci for X-linked sideroblastic anemia.


Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.


Partial gene duplication as a cause of human disease.

Tandem duplication of large regions of DNA, including duplication of whole genes, provides a substrate for genetic evolution. Tandem duplication of smaller regions involving parts of genes is now recognized as a contributor to the mutation spectrum that results in genetic disease. In this review, more than 30 unrelated partial gene duplications that have been implicated in the genesis of human genetic disease are presented and the pathogenic effects and frequency of such duplications are summarized. The mechanisms of duplication formation are analyzed with special emphasis on the molecular details of the nucleotide sequences at the duplication junctions. Evidence to date suggests that duplication may arise from either homologous (Alu-Alu) recombination or nonhomologous recombination, the latter possibly mediated by topoisomerases. For the dystrophin gene, in which most duplications have been identified, these recombination events are intrachromosomal, suggesting that unequal sister chromatid exchange is the major mechanism.


A glycine250--> aspartate substitution in the alpha-subunit of hexosaminidase A causes juvenile-onset Tay-Sachs disease in a Lebanese-Canadian family.

The mutation causing juvenile Tay-Sachs disease (TSD) in two sibs of Lebanese-Maronite origin is described. An mRNA-containing extract of cultured fibroblasts obtained from one of the probands was used as a template to amplify the coding sequence of the hexosaminidase A (Hex A) alpha-subunit. Sequencing of amplified cDNA fragments revealed a single alteration, guanine to adenine at nt 749 creating a G250D mutation. The mutation introduces a new recognition site for the restriction enzyme Eco RV, permitting identification of heterozygotes for this allele following PCR amplification and Eco RV digestion of exon 7 sequences from genomic DNA templates. In order to test the effect of this substitution, an in vitro mutagenized cDNA construct was introduced into a mammalian expression vector and transfected into monkey Cos-1 cells separately or along with a beta-cDNA expression vector. When the mutant alpha-cDNA was the only gene introduced into COS cells no enzymatic activity above endogenous COS cell activity was detected. Cotransfection of normal alpha-cDNA and beta-cDNA followed by immunoprecipitation of human Hex A resulted in 20-fold increase in the ratio between positive and negative (mock transfection) control values. This allowed the detection of some residual activity (12% of the positive control) when the mutant alpha-cDNA replaced its wild-type counterpart. The predicted protein environment in which the mutation occurs is compared to that of the adult-onset Tay-Sachs disease mutation caused by a Gly269-->Ser substitution in exon 7.(ABSTRACT TRUNCATED AT 250 WORDS)


Novel Tay-Sachs disease mutations from China.

We describe three HEXA mutations associated with infantile Tay-Sachs disease (TSD) in three unrelated nonconsanguineous Chinese families. Novel mutations were found in two of these families. The third is a previously reported mutation (G-->A transition at nt 1444) (Nakano et al., 1988). Direct sequencing of PCR products identified a novel insertion of an A after nt 547 in family 1. This change generates an early termination codon 6 bp downstream from the insertion site. Allele-specific oligonucleotide hybridization confirmed homozygosity in the proband. Single strand conformational polymorphism analysis and direct sequencing of amplified exon 13 revealed a T-->C transition at nt 1453 with the corresponding amino acid substitution W485R in the second family. This mutation creates an Fnu4HI restriction site. The proband is homozygous for this allele. When the site-specific mutagenized alpha cDNA carrying the T-->C transition at nt 1453 was expressed in COS 1 cells hexosaminidase S activity was not detectable above background. A G-->A transition at nt 1444 (exon 13) corresponding to the E482K substitution was found in the third family. This mutation occurs at a CpG dinucleotide. It has been reported in an Italian TSD proband and causes defective intracellular transport of the alpha-subunit from the rough endoplasmic reticulum to the Golgi apparatus.


Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a mutation in the COL1A2 gene of type I collagen. The mosaic parent exhibits phenotypic features of a mild form of the disease.

We have determined that a man, ascertained because he fathered a child with lethal osteogenesis imperfecta (OI) with each of two partners, is mosaic in both his germline and somatic tissues for a mutation in the COL1A2 gene which encodes the pro alpha 2(I) chain of type I procollagen. His dermal fibroblasts were previously shown to synthesize a population of cysteine-containing alpha 2(I) chains that were posttranslationally overmodified. DNA sequence analysis of COL1A2 cDNAs demonstrated that the cysteine-containing chain resulted from a point mutation (G to T) in the first position of the codon for the glycine at residue 472 of the triple helical domain. Genomic DNA from the one available affected infant contained the mutant and normal COL1A2 alleles in equal proportion. Examination of DNA from several tissues of the father showed that the mutant allele was present in approximately 40% of his sperm, 80% of his lymphocytes, and nearly 100% of his dermal fibroblasts. Despite the high level of mosaicism detected in somatic tissues, the only phenotypic manifestation of OI in the proband was that he was shorter than his unaffected male relatives and had mild dentinogenesis imperfecta. Thermal stability of type I collagen molecules containing the substitution was decreased, but to a lesser extent than for a nonlethal cysteine for glycine substitution at residue 259 of alpha 2(I), indicating that this measure of molecular stability may be of limited use in explaining the pathogenesis of osteogenesis imperfecta.


A common mutation for mucopolysaccharidosis type I associated with a severe Hurler syndrome phenotype.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the glycosidase alpha-L-iduronidase which is required for the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. Patients with MPS-I store these partially degraded glycosaminoglycans in their lysosomes. MPS-I patients have a wide range of clinical presentations, that makes it difficult to predict patient phenotype which is needed for genetic counselling and also impedes the selection and evaluation of patients undergoing therapy such as bone marrow transplantation. We report the presence of a common mutation accounting for 31% of MPS-I alleles in a study of 64 MPS-I patients. The mutation was originally detected by chemical cleavage and then direct PCR sequencing. The mutation is a single base substitution that introduces a stop codon at position 402 (W402X) of the alpha-L-iduronidase protein and is associated with an extremely severe clinical phenotype in homozygotes. Patients who are compound heterozygotes having one allele carrying the W402X mutation have a wide range of clinical phenotypes. Based on polymorphisms within the alpha-L-iduronidase gene, W402X is associated with three different haplotypes, implying that there is more than one origin for the mutation or that intragenic recombination has occurred. W402X introduces a MaeI restriction endonuclease site into MPS-I alleles enabling its simple detection, which should make possible the assessment of the efficacy of bone marrow transplantation in MPS-I patients homozygous for W402X.


Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German Länder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).


Screening for mutations by expressing patient cDNA segments in E. coli: homocystinuria due to cystathionine beta-synthase deficiency.

Deficiency of cystathionine beta-synthase (CBS) causes the most common form of inherited homocystinuria. We developed a simple CBS expression system in E. coli to screen for pathogenic mutations in affected individuals. Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3. Hybrid CBS was expressed in E. coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation. The first study of a pyridoxine-responsive patient using this screen revealed that of the clones which contained either the middle or the 3'-portion of his cDNA, about half were devoid of catalytic activity. Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T833-->C transition (I278T) and for a paternal A-->C transversion in the intron 11 splice acceptor. The latter mutation leads to an in-frame deletion of exon 12 (nt 1224-1358, amino acids W408 to G453). This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7-10 days of harvesting cultured fibroblasts.


A novel beta-globin structural mutant, Hb Brescia (beta 114 Leu-Pro), causing a severe beta-thalassemia intermedia phenotype.

This study describes a patient with a thalassemia intermedia-like phenotype in whom beta-globin gene sequencing detected a novel abnormal hemoglobin (Hb) due to a T-C substitution at codon 114 of the beta-globin gene arising as a de novo mutation. The abnormal variant was designated Hb Brescia after the place of birth of the propositus. Normal sequences were detected at the in trans beta-globin locus. In addition, alpha-globin gene analysis detected a triple alpha-globin locus which was inherited from the father. The T-C change at position 114 of the beta-globin gene results in a leucine to proline substitution (Leu-Pro) in the G-helix. The resulting Hb tetramer is highly unstable and precipitates forming inclusion bodies in the peripheral red blood cells. Moreover, the Leu-Pro substitution interferes negatively with the four alpha 1 beta 1 contact points of the G-helix most likely adversely affecting the alpha beta dimer formation. The very severe phenotype presented by our patient is unusual in a heterozygote for an unstable Hb variant and may be explained by the coinheritance of the triple alpha-globin locus.


Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12.

The genetic defects responsible for most phenylketonuria (PKU) and hyperphenylalaninemia (HPA) cases are located in the phenylalanine hydroxylase (PAH) gene. Approximately 50-60 mutations have been reported in Caucasians and are reflected in a wide range of clinical severities. Most mutations are linked to specific haplotypes, as defined by eight polymorphic restriction sites in the PAH gene. We hypothesized that there is at least one mild mutation linked to haplotype 12 in the Swedish PKU/HPA population, since 7 of 8 patients carrying haplotype 12 had mild HPA. Sequence analysis revealed a C-to-G transversion at the second base of codon 322, resulting in a substitution of glycine for alanine, in four mutant haplotype 12 genes, and a G-to-A transition at the second base of codon 408, resulting in a substitution of glutamine for arginine, in another three mutant haplotype 12 genes. These mutations segregated with mutant haplotype 12 alleles in nuclear families but were not present on normal or other mutant alleles. Both mutations were tested in a eukaryotic expression system in which enzyme activities of different mutant PAH enzymes reflect the relative severities of the mutations, although these in vitro activities cannot be translated directly into in vivo hepatic activities. The A322G mutant PAH had about 75% and the R408Q mutant PAH about 55% of the wild-type PAH enzyme activity. These in vitro activities are the highest reported for mutant PAH enzymes produced in the same expression system.(ABSTRACT TRUNCATED AT 250 WORDS)


In vitro and in vivo correlations for I65T and M1V mutations at the phenylalanine hydroxylase locus.

Mutations at the phenylalanine hydroxylase (PAH) locus are the major cause of hyperphenylalaninemia. We have previously described four mutations (M1V, IVS12nt1, R408W, and S349P) at the PAH locus in French Canadians with ancestry in eastern Quebec. Here we report (1) identification of another mutation, on a haplotype 9 chromosome, which converts codon 65 from isoleucine (ATT) to threonine (ACT), (2) expression analysis of the I65T mutation in COS cells demonstrating 75% loss of both immunoreactive protein and enzyme activity, and (3) expression analysis of the most prevalent PKU allele (M1V) in eastern Quebec, showing nondetectable levels of PAH protein and activity, a finding compatible with a mutation in the translation initiation codon. Homozygosity for M1V and codominant inheritance of I65T/R408W were both associated with classical phenylketonuria.


Illegitimate transcription of phenylalanine hydroxylase for detection of mutations in patients with phenylketonuria.

Illegitimately transcribed phenylalanine hydroxylase mRNA was amplified using the polymerase chain reaction from both fibroblasts and Epstein-Barr virus-transformed lymphocytes. This method was used to study mutations of this gene in patients with phenylketonuria and known point mutations were easily detected. Illegitimate transcription was successful for studying splicing defects and it was found that the previously described mutation which changes G to A at the 5' donor site of intron 7 causes exon 7 to be spliced out.