Mutations in a gene encoding a novel protein tyrosine phosphatase cause progressive myoclonus epilepsy.

Lafora's disease (LD; OMIM 254780) is an autosomal recessive form of progressive myoclonus epilepsy characterized by seizures and cumulative neurological deterioration. Onset occurs during late childhood and usually results in death within ten years of the first symptoms. With few exceptions, patients follow a homogeneous clinical course despite the existence of genetic heterogeneity. Biopsy of various tissues, including brain, revealed characteristic polyglucosan inclusions called Lafora bodies, which suggested LD might be a generalized storage disease. Using a positional cloning approach, we have identified at chromosome 6q24 a novel gene, EPM2A, that encodes a protein with consensus amino acid sequence indicative of a protein tyrosine phosphatase (PTP). mRNA transcripts representing alternatively spliced forms of EPM2A were found in every tissue examined, including brain. Six distinct DNA sequence variations in EPM2A in nine families, and one homozygous microdeletion in another family, have been found to cosegregate with LD. These mutations are predicted to cause deleterious effects in the putative protein product, named laforin, resulting in LD.

Pressure dependence of the radioactive decay constant of beryllium-7.

Diamond anvil presses of a new design were used to compress samples of beryllium-7 oxide to 120, 210, and 270 kilobars. The decay constant for the conversion of beryllium-7 to lithium-7 by electron capture was measured for compressed and uncompressed samples. A least-squares fit of the equation (lambda(c)-lambda)/lambda = KpP to the experimental data, where lambda(C) and lambda are the decay constants of the compressed sample and an uncompressed sample, respectively, and P is pressure, yields a value of (2.2 +/- 0.1) x 10(-5) kbar(-1) for the constant K(p).

2D dosimetry in a proton beam with a scintillating GEM detector.

A two-dimensional position-sensitive dosimetry system based on a scintillating gas detector is being developed for pre-treatment verification of dose distributions in particle therapy. The dosimetry system consists of a chamber filled with an Ar/CF(4) scintillating gas mixture, inside which two gas electron multiplier (GEM) structures are mounted (Seravalli et al 2008b Med. Phys. Biol. 53 4651-65). Photons emitted by the excited Ar/CF(4) gas molecules during the gas multiplication in the GEM holes are detected by a mirror-lens-CCD camera system. The intensity distribution of the measured light spot is proportional to the 2D dose distribution. In this work, we report on the characterization of the scintillating GEM detector in terms of those properties that are of particular importance in relative dose measurements, e.g. response reproducibility, dose dependence, dose rate dependence, spatial and time response, field size dependence, response uniformity. The experiments were performed in a 150 MeV proton beam. We found that the detector response is very stable for measurements performed in succession (sigma = 0.6%) and its response reproducibility over 2 days is about 5%. The detector response was found to be linear with the dose in the range 0.05-19 Gy. No dose rate effects were observed between 1 and 16 Gy min(-1) at the shallow depth of a water phantom and 2 and 38 Gy min(-1) at the Bragg peak depth. No field size effects were observed in the range 120-3850 mm(2). A signal rise and fall time of 2 micros was recorded and a spatial response of

A scintillating gas detector for 2D dose measurements in clinical carbon beams.

A two-dimensional position sensitive dosimetry system based on a scintillating gas detector has been developed for pre-treatment verification of dose distributions in hadron therapy. The dosimetry system consists of a chamber filled with an Ar/CF4 scintillating gas mixture, inside which two cascaded gas electron multipliers (GEMs) are mounted. A GEM is a thin kapton foil with copper cladding structured with a regular pattern of sub-mm holes. The primary electrons, created in the detector's sensitive volume by the incoming beam, drift in an electric field towards the GEMs and undergo gas multiplication in the GEM holes. During this process, photons are emitted by the excited Ar/CF4 gas molecules and detected by a mirror-lens-CCD camera system. Since the amount of emitted light is proportional to the dose deposited in the sensitive volume of the detector by the incoming beam, the intensity distribution of the measured light spot is proportional to the 2D hadron dose distribution. For a measurement of a 3D dose distribution, the scintillating gas detector is mounted at the beam exit side of a water-bellows phantom, whose thickness can be varied in steps. In this work, the energy dependence of the output signal of the scintillating gas detector has been verified in a 250 MeV/u clinical 12C ion beam by means of a depth-dose curve measurement. The underestimation of the measured signal at the Bragg peak depth is only 9% with respect to an air-filled ionization chamber. This is much smaller than the underestimation found for a scintillating Gd2O2S:Tb ('Lanex') screen under the same measurement conditions (43%). Consequently, the scintillating gas detector is a promising device for verifying dose distributions in high LET beams, for example to check hadron therapy treatment plans which comprise beams with different energies.

Characterization of a scintillating GEM detector with low energy x-rays.

A two-dimensional position-sensitive dosimetry system based on a scintillating gas detector is being developed with the aim of using it for pre-treatment verification of dose distributions in charged particle therapy. The dosimetry system consists of a chamber filled with an Ar/CF(4) scintillating gas mixture, inside which two cascaded gas electron multipliers (GEMs) are mounted. A GEM is a thin kapton foil with copper cladding structured with a regular pattern of sub-mm holes. In such a system, light quanta are emitted by the scintillating gas mixture during the electron avalanches in the GEM holes when radiation traverses the detector. The light intensity distribution is proportional to the energy deposited in the detector's sensitive volume by the beam. In the present work, we investigated the optimization of the scintillating GEM detector light yield. The light quanta are detected by means of a CCD camera or a photomultiplier tube coupled to a monochromator. The GEM charge signal is measured simultaneously. We have found that with 60 microm diameter double conical GEM holes, a brighter light signal and a higher electric signal are obtained than with 80 microm diameter holes. With an Ar + 8% CF(4) volume concentration, the highest voltage across the GEMs and the largest light and electric signals were reached. Moreover, we have found that the emission spectrum of Ar/CF(4) is independent of (1) the voltages applied across the GEMs, (2) the x-ray beam intensity and (3) the GEM hole diameter. On the other hand, the ratio of Ar to CF(4) peaks in the spectrum changes when the concentration of the latter gas is varied.

Loss of heterozygosity and reduced expression of the CUTL1 gene in uterine leiomyomas.

Cytogenetic analyses has revealed deletions and/or rearrangments at several chromosomal positions in approximately half of uterine leiomyomas. The most frequent genetic alteration, deletion of 7q22, was found in approximately 35% of studied cases with cytogenetic abnormalities (128/366=35%). The same chromosomal band was also found to be deleted in a fraction of acute myeloid leukemias and myelodysplastic syndromes. The frequent deletion of 7q22 in some tumors suggest that a tumor suppressor gene may be located in this region. The human Cut-like homeobox gene, CUTL1, is one of the genes localized to 7q22 and it was shown previously to encode a transcriptional repressor that down-modulates the expression of c-Myc. Activation of the c-Myc oncogenic potential has been shown in many cancers to result from alterations in one or the other of its several mechanisms of regulation. These observations led us to hypothesize that CUTL1 could act as a tumor suppressor gene. In the present study, we have identified polymorphic markers within and directly adjacent to CUTL1 at 7q22 and demonstrated that these markers are present in a commonly deleted region in seven out of 50 uterine leiomyomas samples examined. Furthermore, Northern blot analysis revealed that CUTL1 mRNA levels were reduced in eight tumors out of 13. These results suggest that CUTL1 may act as a tumor suppressor gene whose inactivation could be of pathological importance in the etiology of uterine leiomyomas.

Molecular cloning, expression and physical mapping of the human methionine synthase reductase gene.

Methionine synthase reductase (EC 2.1.1.135) is a flavoprotein essential for maintenance of methionine synthase in an active state. We characterized the human gene for methionine synthase reductase (MTRR). The gene is approximately 34kb and comprises 15 exons, varying in size from 43 to 1213bp, and 14 introns whose sizes vary from 108bp to 5kb. The positions of several junctions are conserved between the MTRR gene and the C. elegans ortholog, as well as with the rat cytochrome P450 reductase gene. A 1.3kb CpG island encompasses the 5'-flanking region and exon 1 and extends into intron 1. A short region including the transcription start site is sufficient to confer promoter activity, with a better outcome when accompanied by intron 1. The promoter region contains putative binding sites for Sp1, AP-1, AP-2 as well as CAAT motifs, but no consensus TATA box. Primer extension analysis revealed a single major transcription start site, located 137bp upstream of the previously reported initiator ATG. An alternative splicing event involving a portion of exon 1 predicts that translation can potentially be initiated at two different ATG codons. The gene was physically assigned to a narrow area between markers WI1755 and D5S1957.

Human PON2 gene at 7q21.3: cloning, multiple mRNA forms, and missense polymorphisms in the coding sequence.

We report the cloning and characterization of human PON2, a paraoxonase-related gene-2 that is physically linked with PON1 and PON3 on 7q2l.3. PON2 is ubiquitously expressed and we identified several mRNA forms produced by alternative splicing, or by the use of a second transcription start site. We also describe two polymorphisms in the coding sequences that, in the protein deduced from the longest open reading frame, predict an alanine-to-glycine substitution at residue 147 and a serine-to-cysteine substitution at residue 310.

Manganese-52m, a new short-lived, generator-produced radionuclide: a potential tracer for positron tomography.

A new generator system has been developed using the Fe-52 leads to Mn-52m parent-daughter pair. Fe-52, half-life 8.3 hr, is isolated on an anion-exchange column, and Mn-52m is eluted in hydrochloric acid. Breakthrough is less than 0.01% and the yield is 75%. The 21.1-min half life of Mn-52m is ideal for use in sequential studies, but is long enough to permit radiochemical manipulations to control biodistribution. Animal studies indicate that Mn-52m is an ideal nuclide for myocardial imaging, combining rapid blood clearance and high concentration in the myocardium. An added advantage is that Mn-52m decays 98% by positron emission and is useful for positron computer tomography.

Physical mapping of the chromosome 7 breakpoint region in an SLOS patient with t(7;20) (q32.1;q13.2).

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by multiple congenital anomalies and mental retardation. SLOS has an associated defect in cholesterol biosynthesis, but the molecular genetic basis of this condition has not yet been elucidated. Previously our group reported a patient with a de novo balanced translocation [t(7;20)(q32.1;q13.2)] fitting the clinical and biochemical profile of SLOS. Employing fluorescence in situ hybridization (FISH), a 1.8 Mb chromosome 7-specific yeast artificial chromosome (YAC) was identified which spanned the translocation breakpoint in the reported patient. The following is an update of the on-going pursuit to physically and genetically map the region further, as well as the establishment of candidate genes in the 7q32.1 breakpoint region.

Evaluation of gravimetric assays of the H2O concentration in human serum and urine.

The oven-drying method and the combined freeze- and oven-drying method for gravimetrical measurement of human serum and urine H2O concentration, as well as two reported formulas for the calculation of the serum H2O concentration were evaluated. Day-to-day precision in serum and urine (coefficient of variation (CV) less than 0.95%), and recovery in serum (95-99%) were excellent. Storage at 4 degrees C and at -20 degrees C was safe at least for 3 wk and 2 mth, respectively. For the oven-drying method, which was the most practical, reference values after fasting overnight were determined (n = 47; 99% confidence interval; serum, 48.8-51.6 mol/l; urine, 51.2-53.8 mol/l). Patients in different disease categories were tested (n = 38), and had normal values mostly. Low serum values were found in a patient after hemodialysis with ultrafiltration (47.0 mol/l), and in two patients with an extreme hyperproteinemia (48.8 mol/l) and hypercholesterolemia (48.1 mol/l), respectively. Formulas for calculation of the serum H2O concentration proved unreliable. When direct measurement is impossible, a serum value of 50.5 mol/l can be substituted.

Arterial ammonia with Blood Ammonia Checker II and with indophenol reaction to assess presence of hepatic encephalopathy.

Hepatic encephalopathy (HE) is associated with elevated arterial ammonia levels. The relationship is variable, in part due to ammonia methodology. One method, based on the indophenol reaction (IPh), is interfered with a number of amino acids including all aromatic amino acids. We have determined arterial ammonia simultaneously with the Blood Ammonia Checker II (BAC) as reference method and with the IPh method. The difference BAC-IPh, mumol/l, was assumed to express the interference in the indophenol method (IFI) by amino acids. It may be positive or negative. The aim was to establish the value of BAC in comparison with IPh in the diagnosis of liver disease and overt HE and to assess any added value of IFI. Of two reference groups without disturbances, A (n = 39) had not and B (n = 13) had encephalopathy. Group C consisted of 125 liver patients (34 no cirrhosis, 91 cirrhosis) of which 55 had no manifest HE (C:HE-) and 70 had HE (C:HE+). Median BAC ammonia nitrogen (NH3-N), mumol/l: A 21, B 35, C 80, C:HE - 57 and C:HE+ 98 (A < B < C and A < B < C:HE - < C:HE +, P < 0.001). Median IPh NH3-N, mumol/l: A 27, B 30, C 30, C:HE - 25 and C:HE + 35 mumol/l (A = B = C and C:HE - < C:HE+, P < 0.01). IFI medians: A -6, B 3, C 40, C:HE - 29 and C:HE + 58 mumol/l (A < B (P < 0.05) < C (P < 0.0001); A, B < C:HE - and C:HE+; C:HE- < C:HE + (all P < 0.0001)). While BAC correlated weakly with IPh in the (sub)groups C, C:HE-, C:HE+ (r = 0.3, 0.3, 0.4, P < 0.05), it correlated strongly with IFI (r = 0.9, 0.9, 0.8, P < 0.0001). There was no correlation between IPh and IFI. BAC, as well as IFI, could discriminate all liver patients (C) from both reference groups A and B with 100% positive likelihoods. BAC, IPh and IFI could discriminate between HE- and HE+. To differentiate cirrhosis from non-cirrhosis the specificity of IPh was uniformly high and the sensitivity satisfactory, whereas BAC had a high sensitivity but an insufficient specificity. In conclusion, in blood, BAC is the ammonia determination of choice. It differentiates between reference groups (encephalopathic or not) and liver disease and the more so HE. The combination of BAC and IPh (indicating IFI) may eventually be shown useful to rapidly assess the severity of underlying liver disease in HE patients. In other biological fluids, IPh is excellent when the inhibiting influence of non-protein nitrogen substances is absent or can be eliminated.

Gravimetric determination of the water concentration in whole blood, plasma and erythrocytes and correlations with hematological and clinicochemical parameters.

We have assessed gravimetric methods for determination of intravascular water, established whole blood-, plasma- and erythrocyte water reference values in a healthy volunteer group (n = 97, 48 females) and correlated these variables with 30 simultaneous hematological, clinicochemical and body parameters. The water standard was 55.56 mol/kg = 100 mass %. For erythrocyte water determination three methods were evaluated: 2 indirect methods were easy to perform, the third, using a hematocrit centrifuge, was the most reliable. Imprecision (within-batch coefficient of variation (CV), %) was excellent: whole blood 0.2, plasma 0.1, erythrocytes 0.7-2.2 and recoveries (means, %) 99.7-100.1. Serum water was found to be slightly higher than plasma water. Volunteer group, mean reference values, mass %: whole blood water 79.7, plasma water 91.2, erythrocyte water, three methods 66.2, 64.6 and 64.2, respectively. Females had mean 1.6 mass % higher whole blood water and 0.9-1.0 mass % higher erythrocyte water than males with no difference in plasma water. In the volunteer group whole blood water correlated strongly with hematocrit (r = -0.96), hemoglobin (r = -0.94) and erythrocytes (r = -0.85) and centrifuge hematocrit (r = -0.91). Plasma water correlated strongly with plasma total protein (r = -0.74, all correlations P < 0.001). Hemoglobin and hematocrit can serve as surrogate parameters for whole blood water when water determination is not available; total protein reflects plasma water.

Determination of ammonia in ear-lobe capillary blood is an alternative to arterial blood ammonia.

Blood ammonia determination is a laboratory test to diagnose hepatic encephalopathy. Arterial blood is superior to peripheral venous blood ammonia because of ammonia metabolism in muscle. We have compared capillary with arterial whole blood ammonia as capillary sampling is an attractive alternative. Ear-lobe capillary blood ammonia (ECA) was determined in all 173 persons studied, fingertip capillary blood ammonia (FCA) in 46 of these and arterial blood ammonia (AA) in 113. Of the 173, 60 were healthy (H), 64 were patients, not liver diseased (NLD) and 49 had liver disease (LD). Reference values, median and ranges, mumol NH3-N/l: AA, NLD, n = 64: 17 (7-42); ECA, H = NLD (P = 0.9), n = 124: 20 (7-45); FCA, H = NLD (P = 0.8), n = 33: 70 (29-151). Within the NLD group (n = 64) AA values (range 7-42) were little but significantly lower than the ECA values (range 7-45, P = 0.002). FCA NLD > AA NLD (n = 14, P < 0.0001); FCA H+NLD > ECA (n = 33, P < 0.0001). AA correlated very well with ECA, r = 0.87 (n = 113, P < 0.0001) and less well with FCA, r = 0.56 (n = 27, P < 0.01). ECA correlated with FCA, r = 0.51 (n = 46, P < 0.001). Ear-lobe capillary blood ammonia thus accurately reflects arterial ammonia and is an attractive alternative. The higher fingertip ammonia may be due to contamination with ammonia-rich sweat from finger grooves, regardless of the precautions taken.

Whole blood and plasma water in health and disease: longitudinal and transverse observations and correlations with several different hematological and clinicochemical parameters.

In a healthy reference population, hemoglobin (Hgb) and hematocrit (Hct) have been proposed as surrogate markers for whole blood water (WBW). We have extended this study under different physiological and pathological conditions in two longitudinal series, viz. (1) acute hyper- and hypohydration experiments in a healthy individual and (2) three athletes running 5 km each, and in three transverse series, viz. (3) a young reference population (n = 97, 49 females), (4) an old reference population (n = 37, nine females) consisting of inhabitants of a nursing home and (5) cardiac, hematological and renal patients including severe anaemia, polycythaemia and abnormal protein levels (n = 50, 25 females) with suspected hydration disturbances. The only sex difference found was a lower WBW in males in the young reference group. The percentage change of PW was less than that of WBW. In all five groups together (n = 293) WBW correlated closely (P < 0.0001) with Hgb and Hct (both r = -0.95) and with erythrocyte count (r = -0.85), whereas PW correlated with total protein (Tprot) (r = -0.84). In the longitudinally studied groups (1) and (2) WBW also correlated (P < 0.0001) with cholesterol, Ca, Tprot, albumin, platelets, globulin and white blood cells (r +/- 0.98-0.37), while PW correlated (P < 0.0001) not only with the same clinicochemical parameters but also with Hct, Hgb and red blood cells (r +/- 0.98-0.44). The homeostasis of PW is more narrowly regulated than that of WBW. Hgb, Hct and erythrocyte count reflect WBW and Tprot reflects PW also under disease conditions. WBW (mass%) can be calculated from Hgb and Hct using the formulae: -0.09 x Hgb (g/l) + 91.7 and -28.6 x Hct (v/v) + 91.8 and PW (mass%) from Tprot using the formula: -0.09 x Tprot (g/l) + 97.6. Other correlations were observed only in a longitudinal setting and presumably are due to concentration and dilution.

Comparison of the precision of seven analytical methods for the H2O concentration in human serum and urine.

In order to calculate a true renal H2O clearance (U X V/P), serum and urine H2O concentrations have to be known. In this investigation we compared the precision (repeatability) and the ease of performance of 7 H2O assays in human serum and urine. The 3 gravimetric assays (oven-drying, freeze-drying or freeze-drying as well as oven-drying) had a very high precision (coefficients of variation (CV) 0.2-0.4%) and were easy to perform. The precision of mass spectrometry, gas chromatography and titrimetry (Karl Fischer) was better in urine than in serum (ranges of CV 1.2-1.5% in urine vs. 2.4-4.3% in serum), but the precision of osmometry was better in serum than in urine (CV 1.0 vs. 1.6%). Accuracy was not determined as storage effects at 4 degrees C and at -20 degrees C caused insuperable logistic problems. Only small sample volumes are used in titrimetry and gas chromatography, making them more suitable for determinations in babies and animal studies. With titrimetry determinations can be done in a short time. The gravimetric assays appear to reflect the true H2O content of serum and urine, thus enabling calculation of the true renal H2O clearance, which can be of clinical importance in liver, renal and cardiac disease.

Determination of blood ammonia using the Ammonia Checker.

A new diffusion method employing bromocresol green for determination of blood ammonia (Ammonia CheckerR) using disposable reagent test-plates and a pocket-size colorimeter with direct read-out of results was compared with an enzymatic method. The values obtained with the Ammonia Checker were slightly lower than those with the enzymatic method. Instead of arterial blood, capillary blood may be used for ammonia determination, but thorough cleansing of the fingertip used is necessary because of the high ammonia content of sweat.

Composition of human hepatic bile.

To define reference values of human hepatic bile for sodium, potassium, chloride, calcium, iron, copper, urea, creatinine, phosphate, glucose, bilirubin, cholesterol, protein, bile salts, phospholipids, ammonia, pH, PCO2, bicarbonate and osmolarity, bile was obtained via a T-drain from 12 adult patients who underwent cholecystectomy. Bile of females had a higher cholesterol concentration than that of males. The saturation index, however, was not different in both groups.

Treatment of minor head injuries.

The results of various forms of treatment of patients with minor head injuries have been examined. The number and frequency of post-concussional sequelae was markedly reduced by treatment which included information, explanation and encouragement. Dizziness, loss of hearing and loss of balance were related to external lesions to the vestibular system caused by injuries to the parieto-temporal region of the skull. PTA proved to be a reliable indicator of the severity of cerebral concussion and correlated with post-concussional sequelae and periods of disability. Other factors which increased the number and frequency of post-traumatic sequelae, especially those related to stress before the accident, could be counteracted by better treatment of the patient. The results bear out the hypothesis that post-concussional sequelae start off on an organic basis (PTA) and that persistent sequelae after minor head injuries are also caused by psychogenic, and especially by iatrogenic factors.