Acetazolamide-Responsive Episodic Ataxia Linked to Novel Splice Site Variant in FGF14 Gene.

Here we describe the case of a patient with episodic dizziness and gait imbalance for 7 years and a negative family history. On clinical examination, interictally, the patient presented with gaze-evoked nystagmus and rebound nystagmus and slight dysarthria. MRI of the brain was normal and peripheral-vestibular function was bilaterally intact. Based on genetic testing (episodic ataxia panel), a heterozygote splice site variant in intron 1 of the FGF14 gene was identified. This report adds important new evidence to previous observations that pathogenic variants in the FGF14 gene may result in variable phenotypes, either in progressive spinocerebellar ataxia (type 27) or in episodic ataxia as in our case. Our patient responded well to acetazolamide (reduction in the frequency of attacks by about two thirds), supporting the hypothesis of a sodium channelopathy.

Severe phenotype with cis-acting heterozygous PMP22 mutations.

We report on a 20-year-old male with severe Charcot-Marie-Tooth (CMT) disease and a de novo deletion (c.281delG, p.G94AfsX17) on the paternal PMP22 allele harboring c.353C>T (p.T118M). RNA-based sequence analysis confirmed the absence of nonsense-mediated decay and the presence of the mutant transcripts in Epstein-Barr virus-transformed lymphoblastoid cells of our patient. His clinical findings included early onset of polyneuropathy, loss of muscle mass with distal pareses, hammer toes, and progressive scoliosis. There was no neuropsychological alteration. Our results suggest that the deletion c.281delG alone is responsible for the severe CMT phenotype. To the best of our knowledge, this is the second report on a proven paternal origin of a de novo single-base mutation in the PMP22 gene.

Becker muscular dystrophy with marked divergence between clinical and molecular genetic findings: case series.

Both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations of the X-linked dystrophin gene. BMD patients are less affected clinically than DMD patients. We present five patients with a diagnosis of BMD. First, two identical twins, with a deletion of exon 48 of the dystrophin gene, who experienced prominent muscle cramps from the age of three. The histopathological examination of muscle biopsies of these two twins revealed only very slight muscle fiber alterations. Second, two brothers who displayed marked, unusual intrafamilial variability of the clinical picture as well as showing a new point mutation in the dystrophin gene. And finally, a fifth boy who displayed a new point mutation in the dystrophin gene. Although he was clinically asymptomatic at the age of 15 and muscle biopsy only showed very minor myopathic signs, serum Creatine Kinase (CK) levels had been considerably elevated for years. Taken together, these cases add to the spectrum of marked discrepancies in clinical, histopathological and molecular genetic findings in BMD.

The location of constitutional neurofibromatosis 2 (NF2) splice site mutations is associated with the severity of NF2.

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1-5 had more severe disease than those with splice site mutations in exons 11-15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours.

Assessment of P-glycoprotein, glutathione-based detoxifying enzymes and O6-alkylguanine-DNA alkyltransferase as potential indicators of constitutive drug resistance in human colorectal tumors.

Drug resistance is a major problem in cancer chemotherapy. Treatment protocols generally include a number of different cytotoxic drugs given in combination. Therefore, drug resistance in the tumor is likely to result from the coexpression of several cellular activities able to prevent cell killing by any of the drugs used. In this study we have measured several potential drug resistance mechanisms consisting of the multidrug resistance gene product P-glycoprotein, glutathione, glutathione-transferase and -peroxidase, and the DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase in samples of colon carcinoma and normal adjacent mucosa from 23 untreated patients. All of these, with the exception of P-glycoprotein, showed significant increases in tumor tissue levels when compared with normal tissue from the same patient. The significance was highest for glutathione peroxidase (P less than or equal to 0.0005). Individual patients, however, showed very different patterns, with none, several, or all monitored resistance mechanisms elevated in the tumor. The implications both in the choice of drugs and in the use of resistance modifying agents to improve therapy for the individual patient are discussed.

Type of inducing signal regulates transactivation by p53.

The tumor suppressor gene p53 is expressed in the contrasting cell fates apoptosis and proliferation. We examined whether the transactivation of the p53 target genes, waf1 and mdm2, is dependent on the cause of p53 induction in human peripheral blood mononuclear cells (PBMC). Both apoptosis triggered by the purine analog 2-chlorodeoxyadenosine (CdA) and growth stimulation by the mitogen phytohemagglutinin (PHA) induced a comparable level and time course of p53 mRNA expression. Both stimuli led also to an increase of p53 protein levels. The cytotoxic agent, but not the mitogen, led to transactivation of waf1 and mdm2 within 18 h. Transactivation was followed by apoptosis of 89% of the PBMC within 48 h. The c-myc oncogene and poly(ADP-ribose)polymerase (PARP), which also have a dual function in proliferation and apoptosis, showed an early induction by both CdA and PHA. These results add further evidence that growth stimulation and DNA damage-induced apoptosis share early gene activation pathways in normal cells. However, since p53 does selectively translate into transactivation of target genes depending on the cause of induction, this function of p53 seems to be regulated by additional factors, which are closely related to the ultimate fate of the cell.

Human lymphocyte proliferation. I. Correlation between activated and proliferating T-lymphocytes.

The response of human peripheral blood lymphocytes to Con A and PHA has been analyzed by [3H]thymidine incorporation and cytofluorometry. Using the latter method, it is possible to quantitate the number of cells in the G0 phase (normal RNA and DNA content) and in the G1 phase (elevated RNA, but normal DNA content). A very high correlation is found between numbers of Con A or PHA-induced G1 cells and [3H]thymidine incorporation in healthy donors. This high correlation is found when culture medium is enriched with 10% autologous plasma or 10% AB-serum. The use of a recently developed defined serum-free medium (RPMI 1640 with albumin, alanine, transferrin, sodium selenite and zinc chloride), however, suggest that donors can be divided into two groups according to different medium requirements for PHA-stimulated lymphocytes. Because several immunoregulatory mechanisms at the level of T-lymphocytes take place in the G1 phase, it can therefore be expected that cytofluorometric analyses of lymphocytes in the various cell cycle phases may improve the interpretation of altered lymphocyte response to lectins and antigens.

Aging and immunity: decrease in interleukin-2 production and interleukin-2-dependent RNA-synthesis in lectin-stimulated and murine spleen cells.

The RNA-content of G1 cells in lectin-stimulated spleen cell cultures of young and aged NMRI mice was determined by flow cytometry. In spleen cells of aged mice a preferential decrease of G1 cells with a high RNA-content, so-called G1b cells, was found. Since, as shown in a previous report, only cells with a high RNA-content are able to proliferate and the passage of low (G1a) to high (G1b) RNA-content is interleukin-2(IL-2)-dependent, the ability of young and old spleen cells to produce IL-2 was tested. In old spleen cells a diminished production of IL-2 was found. Addition of external IL-2, however, did not increase the proliferative capacity of old spleen cells, nor did it induce more G1b cells. Thus spleens of aged mice contain cells, which can be activated by lectin, but then fail to respond to IL-2. Both decrease in IL-2 production and receptivity for IL-2 may contribute to the diminishing immune response in aging individuals.

Age-related changes of mitogen responsiveness in different lymphoid organs from outbred NMRI mice.

Lymphocytes from spleen, thymus and lymph nodes from individual young adult (3--4 months) and aged (26--30 months) NMRI mice were stimulated with the mitogens Con A, PHA and LPS. 24 hours later, the number of cell with increased RNA-content (G1 cells) was determined by cytofluorometry. In parallel the 3H-thymidine incorporation after 48 hours was measured for the same cell samples. Aged animals in average produced less G1 cells and incorporated less 3H-thymidine as compared to young adults. By calculating the 3H-thymidine incorporation per G1 cell, the proliferative capacity of mitogen-induced G1 cells can be estimated. These ratios are lower in aged mice as compared to young adult, suggesting that in these animals not only less cells can be activated as measured by cytofluorometry, but also from these activated cells again fewer continue the cell cycle by initiating DNA-synthesis. In response to Con A and PHA, aged mice in average produce less G1 cells in all of the three lymphoid organs tested. In response to LPS, however, the young adult produced only few G1 cells in lymph nodes and practically none in thymus, whereas in aged animals a considerable number of G1 cells was found in both organs. Corresponding results were found for the 3H-thymidine incorporation. These results indicate that in addition to the reduction of the mitogen-response an age-related change in the distribution of mitogen-responsive cells in the different lymphoid organs takes place.

Patterns of drug resistance parameters in adult leukemia.

P-glycoprotein (Pgp), Glutathione (GSH), Glutathione S-Transferase (GST), and O6-Alkylguanine-DNA Alkyltransferase (ATase) were measured in parallel as putative indicators of drug resistance in adult leukemia. The patterns of resistance parameter expression of chronic and acute leukemia were different. In acute leukemia on average all parameters were increased as compared to normal bone marrow. In chronic leukemia GSH and GST were increased, whereas Atase, GPx and frequency of Pgp-expression were low. Treatment with cytostatic drugs did not influence median levels of expression/activity of the resistance parameters. Resistance parameter expression/activity of leukemic cells was also compared with various other tissue and tumor types. Generally the pattern of resistance parameter expression reflected the resistance status of the tissue, constitutively resistant tumor types and their corresponding normal tissue on average having higher levels than leukemic cells and other tissue and tumor types with acquired resistance. For individual patients with acute leukemia, however, none of the parameters was directly correlated with response to treatment.

Similarity of apoptosis induction by 2-chlorodeoxyadenosine and cisplatin in human mononuclear blood cells.

The purine analogue 2-chlorodeoxyadenosine (CdA) is unique compared with traditional antimetabolite drugs, as it has shown equal activity in dividing and resting lymphocytes. Poly(ADP-ribose)polymerase (PARP) activation and consecutive NAD+ consumption have been associated with the induction of apoptosis in resting cells. The potential of CdA to induce the p53-dependent DNA damage response was assessed in resting and phytohaemagglutinine (PHA)-activated peripheral blood mononuclear cells (PBMCs) and compared with cisplatin (DDP), a cell cycle-dependent and DNA-damaging agent that is mainly used in the treatment of solid tumours. Both drugs induced transactivation of the p53 target genes waf1 and mdm2, NAD+ consumption and apoptotic death. The expression pattern of p53 and waf1 suggests a partly p53-independent induction of waf1. The expression of c-myc and PARP, which both have a dual role in proliferation and apoptosis, was selectively induced by CdA. Cell cycle stimulation increased the cytotoxic activity of both drugs. These data show that DDP is also a potent inducer of apoptosis in resting and proliferating peripheral blood mononuclear cells. Activation of the p53-dependent DNA damage response seems to be an important component of the toxic effect of CdA.

Age-related changes in g0-g1 transition and proliferative capacity of mitogen-stimulated murine spleen cells.

The early kinetics of mitogen-stimulated of mitogen-stimulated spleen cells from young adult and aged NMRI mice were investigated. Using RNA synthesis as measured by cytofluorometry by cytofluorometry and 3H-leucine and 3H-thymidine incorporation as parameters, it was apparent that age-related changes occur at different levels during mitogen stimulation. First, in aged mice fewer cells per 10(6) spleen cells are being activated by mitogen, and, second, fewer of the activated lymphocytes will proliferate. However, the cells which initiate synthesis of RNA, protein, and DNA apparently behave in identical manners, chronologically, as cells from young adult mice.

Ageing and immunity in outbred NMRI mice: lack of correlation between age-related decline of the response to T cell mitogens, the antibody response to a T-dependent antigen and lifespan in outbred NMRI mice.

Responses to different mitogens and the primary in vitro antibody response to a T-dependent antigen (SRBC) were tested in individual mice of various ages from an outbred strain (NMRI). Comparing mean values, an age-related decrease was found only for the responses to Con A and PHA but not for LPS nor for the primary in vitro antibody response to SRBC. The magnitude of the responses obtained in individual mice of the same age varied greatly. Some of the young as well as aged animals could respond very well to antigen, mitogen or both and there was a complete lack of correlation between the magnitude of individual responses to mitogen and the number of PFC produced in the in vitro antibody response to SRBC. Additionally, we investigated whether the magnitude of the immune responses measured in individual mice had any influence on their lifespan. The mitogen response and the in vitro PFC production of spleen cells from individual semi-splenectomized mice were therefore compared with their lifespan. The lifespan did not appear to be influenced by any of the immune responses measured.

The influence of serum on lymphocyte cultures. II. Cell cycle specificity of serum action in spleen cells.

Serum exerts several effects in lymphocyte cultures, one of them being manifested very early. The presence of fetal calf serum (FCS) results in a markedly higher thymidine uptake within a few minutes, as compared with serum-free cultures. On the other hand, the uptake of uridine and other purine bases seem to be little influences by serum. Experiments comparing the uptake of thymidine into the cytoplasm or into trichloroacetic acid-precipitable material suggested that the intracellular thymidine pool increases in size when FCS is added. Using a Lineweaver--Burk plot for thymidine and uridine uptake over a 4-h period, no changes in uridine uptake were observed in the presence of FCS; on the contrary, serum induced an increased Vmax for thymidine, whereas Km remained constant. Cytofluorographic quantitation of G0 and G1 cells indicated that cells disappear more rapidly from the G1 phase in the presence of FCS. The addition of hydroxyurea to the cultures prevented this disappearance. The results taken together strongly suggest that serum contains a factor promoting the shift of G1 cells into the S phase. When a 4-h culture period was used, all sera from different species tested at low concentrations appeared to contain this activity.

Age-related changes in the formation of glucocorticoid and insulin receptors during lectin-induced activation of human peripheral blood lymphocytes.

The influence of age on the number of receptors for insulin and glucocorticoids on human T cells was examined. The specific binding of 125I-insulin and 3H-dexamethasone to phytohemagglutinin-(PHA)-stimulated T cells was found to decrease with age. Populations of PHA-stimulated T cells, however, are heterogeneous with respect to cell cycle phases, and cells in different cell cycle phases have been shown to bear different numbers of receptors. Therefore, it was not clear whether the measured decrease in specific binding in cultures from aged donors was due to a decreased number of activated cells or to a decreased number of binding sites per cell. In parallel to measurements of receptors, performed at different times following stimulation (20 and 44 h), numbers of cells in the different early cell cycle phases G0, G1a and G1b were quantitated by flow cytometry. In this way the receptor number per cell in each phase of the cell cycle could be determined. Receptor numbers on resting cells and in the earliest phase of activation (G1a) were found not to be influenced by age. A decreased receptor density, however, was apparent on G1b cells from aged donors.

Breast cancer: pretreatment drug resistance parameters (GSH-system, ATase, P-glycoprotein) in tumor tissue and their correlation with clinical and prognostic characteristics.

BACKGROUND: The identification of new factors predicting relapse, outcome and response to systemic therapy in breast cancer is warranted. The measurement of biological markers such as drug resistance parameters (DRPs), which are part of the phenotype of malignant cells and contribute to resistance to anti-cancer drugs may be a possibility, which may ultimately lead to improvement of therapeutic results. PATIENTS AND METHODS: The level of glutathione (GSH), activities of glutathione-S-transferase (GST), glutathione-peroxidase (GPx), 06-alkylguanine-DNA-alkyltransferase (ATase), and P-glycoprotein (PGP) were measured in tumor and adjacent tumor free tissue samples from 89 consecutive, untreated females with breast cancer and correlated with clinical and prognostic factors. Early breast cancer (EBC) was diagnosed in 56 patients, 22 patients had locally advanced (LABC) and 11 patients metastatic breast cancer. RESULTS: All DRPs showed significantly higher expression in tumor than in tumor free tissues. GPx was positively correlated with GST (r = 0.3, P = 0.0048) and with GSH (r = 0.5, P = 0.0001) in tumor as well as in normal tissue. GST activity was significantly higher in EBC than in LABC or metastatic breast cancer (P = 0.02). GSH level was significantly higher in grade 1 than in grade 2 or grade 3 tumors (P = 0.01). When clinical characteristics were related to the level of DRP, 'high' GSH was associated with age > 60 years (P = 0.01) in EBC, and with grade 1-2 tumors (P = 0.05) in LABC. No differences in OS were apparent between groups of 'high' and 'low' DRP-expression. However, the four-year estimated disease-free survival of EBC tended to be higher in patients with 'high' GST (P = 0.10) and of LABC in patients with 'high' GPx levels (P = 0.06). CONCLUSION: We conclude that 'high' levels of DRP in tumor tissue of breast cancer patients are part of the initial phenotype of the malignant cells. Due to its high prevalence (83% in EBC, 100% in primarily metastatic breast cancer), PGP did not add to prognostic information. High levels of GSH, GST and GPx were associated with favorable clinical characteristics and good prognosis, whereas low levels of GSH and GST activity were associated with more aggressive or more advanced disease.

Clinical and molecular analysis of chinese patients with thyrotoxic periodic paralysis.

Although sporadic thyrotoxic periodic paralysis (TPP) has a much higher prevalence in Asian than in all the other populations studied so far, it is also increasingly being seen at the emergency departments of the West, hence, it is vital to stress the importance of recognizing it. TPP shares some similarities with hypokalemic periodic paralysis (HOKPP). However, the pathophysiology of TPP and the reasons for this higher incidence are not known. We hypothesized that some mutations in the CACNA1S gene, which has been implicated in familial HOKPP, might play a role in TPP. We present 5 Chinese patients who suffer from TPP and demonstrate typical clinical features. No mutation was found on the whole CACNA1S gene. Therefore other molecular mechanisms will have to be examined in order to explain the different TPP incidences.

Multiple drug resistance parameter expression in ovarian cancer.

OBJECTIVE: Drug resistance represents a complex problem for the treatment of ovarian cancer. This study was undertaken to assess several putative resistance parameters (DRP) in parallel in cancer tissue from newly diagnosed patients with ovarian cancer in order to establish possible correlations to known clinical factors and prognosis. MATERIAL AND METHODS: Tumor and adjacent tumor free ovarian tissue samples from 39 consecutive, untreated female patients with ovarian cancer were obtained and as potential DRPs, the level of glutathione (GSH), the activities of glutathione S-transferase (GST), glutathione-peroxidase (GPx), O6-alkylguanine-DNA alkyltransferase (Atase), and topoisomerase II (TOPO) were assessed biochemically, and P-glycoprotein (Pgp) was assessed by Western blotting. RESULTS: Interindividual variations were high and each patient exhibited an individual profile of resistance factor expression. Levels of GSH were increased with stage (linear trend: P < 0.002), and GST, GPx, and Atase showed a similar tendency. With few exceptions no correlation was found between the DRPs and other prognostic characteristics. All tested DRPs except Pgp showed significantly higher levels/activities in tumor tissues than in the surrounding tumor free tissues (P < 0.05). The tested DRPs were found not to influence response to treatment. CONCLUSIONS: It is concluded that elevated DRPs reflect an intrinsic pattern of components of the detoxifying system in the tumor tissue. This pattern differs between patients and may partly explain the difficulty to assess the clinical importance of individual DRPs in order to translate them into recommendations for specific therapies.

Analysis of 3H-histamine interaction with lymphocytes: receptor binding or uptake?

The immunoregulatory role of histamine is presumably mediated by specific receptors on the plasma membrane of lymphocytes. However, using murine spleen cells and a whole cell assay commonly applied in hormone receptor studies, specific histamine receptors with an affinity higher than that of non-specific binding could not be identified. Nevertheless, approximately 30% of the totally bound histamine was undissociable over a range of added histamine concentrations (9 X 10(-6)-1 X 10(-2) M). Lectin stimulation of spleen cells caused an additional two-fold increase of undissociable histamine. The H1 receptor antagonist, diphenhydramine, blocked histamine uptake, whereas the H2 receptor antagonist, cimetidine, had no effect. Binding experiments carried out at 4 degrees C demonstrated that the amount of undissociable histamine was much reduced. Even at 4 degrees C, evidence for specific membrane associated histamine receptor could not be obtained. It was therefore concluded that lymphocytes take up histamine by an energy-dependent mechanism inhibitable by diphenhydramine but not cimetidine, and that the usual hormone receptor methodology did not allow the identification of specific membrane associated histamine receptors.