PMM2 mutation spectrum, including 10 novel mutations, in a large CDG type 1A family material with a focus on Scandinavian families.

Carbohydrate-deficient glycoprotein syndrome type IA (CDG IA) is an autosomal recessive disease characterized clinically by severe involvement of the central and peripheral nervous system, and biochemically by complex defects in carbohydrate residues in a number of serum glycoproteins. CDG IA is caused by mutations in the PMM2 gene located in chromosome region 16p13. In this study, 61 CDG type IA patients (122 chromosomes) were screened for mutations in the PMM2 gene using a combination of SSCP and sequence analysis. More than 95% of the mutations could be detected. All of them were missense mutations. Mutations 422G>A and 357C>A were strikingly more common in the material and comprised 58% of mutations detected. Of the 20 mutations found, 10 were not reported previously. Seven mutations, e.g. 26G>A (five alleles) and 548T>C (seven alleles), were found only in Scandinavian families. The most common genotype was 357C>A/422G>A (36%). Three patients were homozygous, 357C>A/357C>A (two cases), and 548T>C/548T>C (one case). No patients homozygous for the most common mutation 422G>A were detected. The different mutations were clustered e.g., in that most were located in exon 5 (five) and exon 8 (six), while no mutation was detected in exon 2. When the frequencies of each mutation were included, exon 5 comprised 61% (65 chromosomes) of the mutations; in Scandinavian patients the frequency of these mutations was 72%. Thus, analysis of exon five in these patients enables both reliable and time-saving first screening in prenatal diagnostic cases. This could be followed by a second step of additional strategies for the detection of other mutations.

Detailed mapping of the phosphomannomutase 2 (PMM2) gene and mutation detection enable improved analysis for Scandinavian CDG type I families.

The gene for carbohydrate-deficient glycoprotein syndrome type I (CDG1) has previously been localised by us close to marker D16S406 in chromosome region 16p13.2-3. We also presented data indicating a strong founder mutation associated with a specific haplotype in CDG I patients from western Scandinavia. The phosphomannomutase 2 (PMM2) gene was recently put forward as a likely CDG1 candidate gene. We have now shown that the specific haplotype is associated with the PMM2 mutation 357C > A. Using data from radiation hybrid panel we have refined the position of the PMM2 gene to very close to marker D16S3020 in the interval between D16S406 and AFM282ze1 on the distal side and D16S3087 on the proximal side. Due to the severity of the disease many families request prenatal diagnostic services for CDG I. In the meantime, until the mutation spectrum is fully examined, we propose the combined use of mutation analysis and linkage analysis with polymorphic markers as diagnostic tools for Scandinavian CDG I families requesting prenatal diagnosis. Using this strategy we have to date successfully performed 15 prenatal diagnoses for CDG I.

Alcohol abuse increases the lipid structural order in human erythrocyte membranes. A steady-state and time-resolved anisotropy study.

The effect of ethanol abuse on the lipid ordering of the human erythrocyte membranes was studied by steady-state and time-resolved fluorescence anisotropy measurements of DPH and its polar analogue TMA-DPH, which probe different membrane regions. Steady-state anisotropy values with DPH as a probe were slightly but significantly increased (+3%) in erythrocyte membranes from alcoholic patients. A resistance to the ethanol fluidizing effect was evidenced in these membranes with DPH and TMA-DPH. No difference in the probe lifetimes was detected between the control and the alcoholic subjects. In the alcoholic patients as compared to the healthy controls, the residual anisotropy for DPH was significantly increased (+7%) corresponding to an increase in the orientational order parameter of 4%; a decrease of the apparent correlation time value was also observed. Nevertheless, no differences between the two erythrocyte populations were observed with TMA-DPH.

Levels of transforming growth factor alpha (TGF-alpha) in human cerebrospinal fluid.

In this study, we investigated cerebrospinal fluid of patients with various neurological symptoms for the presence of transforming growth factor alpha (TGF-alpha). 41 samples of cerebrospinal fluid were collected by lumbar puncture performed routinely due to the clinical suspicion of neurological disease from 22 females (age 15-80 years, median 42 years) and from 19 males (age 18-82 years, median 48 years). A highly sensitive and specific radioimmunoassay was used to determine the concentration of TGF-alpha in the samples. The detection limit of the assay was about 200 pg TGF-alpha. There was no cross-reactivity to human EGF. We showed CSF indeed does contain TGFalpha. As TGF-alpha was detected in all 41 samples investigated, this growth factor appears to be a constant component of CSF. The mean concentration was 5.5 ng TGF-alpha (S.D. +/- 2.7 pg/ml, range 1.1 to 13.9 pg/ml). There was no significant correlation between TGF-alpha concentration in CSF and age (r = -0.006) and there was no significant difference between females (mean 5.8+/-3.10 pg/ml) and males (mean 5.2+/-1.96 pg/ml). No diagnosis was over represented in patients with TGF-alpha concentrations above or below 1 S.D. off the mean. However, highest concentrations of TGF-alpha were found in the group of patients with peripheral neurological sensory dysfunctions and polyneuropathy. We conclude that TGF-alpha is not only a constant component of human cerebrospinal fluid in adults but could also be significantly involved in the pathophysiology of various neurological diseases. The earlier hypothesis that TGF-alpha could mainly have a role in brain development needs hence to be re-evaluated.

Direct immunofixation after isoelectric focusing. An improved method for identification of cerebrospinal fluid and serum proteins.

An improved method for identification of CSF and serum proteins is described, using analytical isoelectric focusing followed by direct immunofixation in polyacrylamide gel. This method offers high sensitivity together with retained resolution after isoelectric focusing and is technically easy to perform. The gamma-trace protein, normal transferrin, haptoglobin and alpha 2-macroglobulin and two genetic alterations of transferrin were analyzed. The qualitative differences, previously observed on crossed immunoelectrofocusing, between transferrin and alpha 2-macroglobulin in the CSF and serum were confirmed by immunofixation. Protein components, differing in isoelectric pH by at least 0.03 pH unit and 0.5 mm apart were readily identified by this technique.

Crossed immunoelectrofocusing for identification of normal and abnormal cerebrospinal fluid proteins. A preliminary report.

Isoelectric focusing is a valuable method for the analysis of cerebrospinal fluid (CSF) proteins. Its high resolving power has, however, created problems in identifying the large number of protein bands separated. In an attempt to identify these bands, two-dimensional crossed immunoelectrofocusing has been used, where isoelectric focusing is combined with rocket immunoelectrophoresis. By these methods 9 of the normal proteins in the acidic pH interval have been identified. In addition the unusual CSF protein abnormalities occurring in Marie-Sanger-Brown's ataxia and alcoholic cerebellar degeneration have been shown to represent increases of different microheterogeneous forms of transferrin.

The normal cerebrospinal fluid proteins identified by means of thin-layer isoelectric focusing and crossed immunoelectrofocusing.

The cerebrospinal fluid and serum proteins from 32 healthy individuals have been examined by isoelectric focusing in a pH-gradient from 3.5--11.0. Seventeen normal proteins have been identified by means of crossed immunoelectrofocusing. The pI-values of the main fractions of these proteins have been determined. On crossed immunoelectrofocusing microheterogeneity was observed for all of the proteins except haemopexin, gamma-trace protein of CSF and prealbumin of serum. Differences between serum and CSF were observed for 6 of the proteins. Prealbumin of CSF had a lower pI-value than in serum and showed partial immunological identity with prealbumin of serum. Transferrin of CSF included several components with lower sialic acid contents than in serum. The conversion of C'3-complement from beta-1-C to beta 1-A was slower in CSF than in serum. Qualitative differences between CSF and serum were also noticed for alpha2-macroglobulin and haptoglobin. IgM was not detected in CSF. In CSF the gamma-trace protein was found on isoelectric focusing in one third of the subjects. Two subjects had 2 close tau-fractions and another two had one unidentified band in the anodal IgG-area. The gamma-trace protein did not show any proteolytic activity, nor any resemblance in charge to 4 basic enzymes or with the histones tested.

The sialic acid and galactose concentrations in erythrocyte membranes in patients with myotonic dystrophy, limb-girdle and facioscapulohumeral dystrophy.

Sialic acid is an important constituent of membrane-bound glycoproteins and glycolipids. It occurs linked to galactose at the surface of the membrane and is involved in, e.g., cation exchange, receptor function, maintenance of membrane polarity and intercellular interactions. In myotonic dystrophy there is evidence of an as yet basically undefined plasma-membrane abnormality. Considering the importance of sialic acid in membrane function, sialic acid as well as galactose concentrations were measured in erythrocyte membranes from 17 patients with myotonic dystrophy and compared to 17 matched healthy controls. There was a highly significant (P less than 0.0005) reduction of the sialic acid concentration in the patients, while no significant difference in galactose concentration was found. In 16 patients with limb-girdle and facioscapulohumeral dystrophy, sialic acid and galactose concentrations did not differ from matched controls. The possible importance of a reduced concentration of membrane-bound sialic acid in myotonic dystrophy is discussed in relation to previously reported biochemical membrane abnormalities in this disease.

Carbohydrate composition of erythrocyte membranes and glycosidase activities in serum in patients with myotonic dystrophy, limb-girdle dystrophy and congenital myotonia.

A number of abnormalities in cell membrane function, including cells other than muscle cells, have been described in patients with inherited muscular diseases such as myotonic dystrophy and congenital myotonia. The basic molecular defects are, however, still unknown. The complex carbohydrates of membrane-bound glycoconjugates are of vital importance for the normal performance of the cell membrane. In this study the concentrations of the three major carbohydrates (sialic acid, galactose and hexosamines) of the erythrocyte membrane were therefore determined in patients with myotonic dystrophy, limb-girdle dystrophy and congenital myotonia. The activities of relevant glycosidases in serum were also assayed. In each of the three diseases pertinent changes of the carbohydrate pattern were found. In patients with myotonic dystrophy the sialic acid and in patients with limb-girdle dystrophy the hexosamine concentration was significantly reduced (P less than 0.0005). The sialic acid, galactose and hexosamine concentrations were all significantly increased in patients with congenital myotonia. No increase of the neuraminidase (sialidase) activity was found in sera from patients with myotonic dystrophy. In patients with limb-girdle dystrophy, the activities of serum hexosaminidases were normal. These results support the contention that certain inherited muscular diseases may represent generalized membrane disorders, and suggests that disturbances of membrane-bound glycoproteins and/or glycolipids might be of importance in the pathogenesis of some of these disorders.

Isoelectric focusing and electrophoresis of cerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies.

In the very few previous investigations of the CSF-proteins in muscular dystrophies the results have generally been reported as normal. In spinal muscular atrophies a barrier-damage pattern of CSF-proteins has been found in amyotrophic lateral sclerosis (ALS). In the present investigation the CSF-proteins were examined by isoelectric focusing and quantitative paper electrophoresis in 13 patients with muscular dystrophies and in 11 patients with spinal muscular atrophies. On isoelectric focusing, CSF-protein abnormalities were found in 85% of the cases with muscular dystrophies and in all patients with spinal muscular atrophies. Differences in the CSF-protein patterns were observed within the group of muscular dystrophies and between these and the cases of spinal muscular atrophies. In ALS and in myotonic dystrophy, abnormal CSF-protein fractions occurred mainly in the alkaline pH-range, while in limb-girdle dystrophy and the patient with facioscapulohumeral dystrophy, aberrant fractions appeared mainly in the acidic region. CSF-protein abnormalities were found in both the alkaline fractions (HAFs) with pI 9.2-9.6 and a fraction with PI 7.1 were found in half of the patients with myotonic dystrophy. The CSF electrophoresis in myotonic dystrophy showed increased levels of beta1-globulin in all cases examined. Signs of barrier-damage were commonly encountered in ALS in contrast to the muscular dystrophies, except for myotonic dystrophy. The results are discussed in terms of possible diagnostic value and with regard to pathogenetic significance, particularly in relation to the current hypothesis of a neural involvement in muscular disorders.

Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia.

The CSF findings in hereditary ataxias and allief disorders have hitherto mostly been reported as normal if one excludes Refsum's syndrome. The CSF-protein patterns found on isoelectric focusing and quantitative paper electrophoresis were studied in 12 patients with hereditary ataxias and hereditary spastic paraplegia. Using a recently-developed technique of isoelectric focusing of CSF-proteins in flat beds of polyacrylamide gel, the authors could show abnormal CSF-protein patterns in all but 1 of the present cases. The aberrant CSF-protein patterns found showed differences between the syndromes studied. Two unique patterns with conspicuous fractions in the acid range were observed in patients with Marie-Sanger-Brown's ataxia (mother and daughter) and Holmes' ataxia. A third CSF-protein pattern was found in a sibship with Friedreich's ataxia including a double fraction in the acid region (pI 5.9-6.1) in all 4 subjects and a highly alkaline fraction (HAF) with pI about 9.3, in 3 of them. Similar acid fractions (pI 5.9-6.1) were also detected in 3 of 4 patients with hereditary spastic paraplegia, a brother and sister showing a very similar CSF-protein pattern. Double fractions with pI 5.9-6.1 and/or HAF may also occur in other neurological diseases, mostly, however, associated with other distinctive features of their CSF-protein patterns. A possibility in the future of distinguishing hereditary CNS-diseases by examination of the CSF-protein pattern is suggested.

Isoelectric focusing and electrophoresis of the CSF proteins in tremor of different origins.

The CSF proteins have previously been very little investigated in the cerebellar syndrome of chronic alcoholism and in essential tremor. Such studies have been carried out more thoroughly by electrophoretic methods in Parkinson's disease but generally with normal results. In the present investigation the CSF proteins were examined by isoelectric focusing and quantitative paper electrophoresis in 10 patients with the cerebellar syndrome of chronic alcoholsm, 12 patients with Parkinson's disease and 16 subjects with essential tremor. Abnormal CSF proteins of very similar appearance were found on isoelectric focusing in the acidic pH interval 5.6-5.8 in 80% of the patients with the cerebellar syndrome of chronic alcoholism. In Parkinson's disease the most common aberration was evidence of nonspecific blood-CSF-barrier damage which occurred in half of the patients. In only 17% of these cases did other alterations appear, situated in the pH range alkaline to pH 5.8. Abnormal CSF proteins were found in 94% of the patients with essential tremor. The aberrant proteins appeared in both the acidic and alkaline pH regions, most frequently with anisoelectric point at pH 5.9, 7.2 and 9.3. There was a considerably higher frequency of CSF protein abnormalities in different pH ranges in patients with tremor of more pronounced degree as compared to those with only mild symptoms. The electrophoretic examinations failed to show any conclusive alterations. Barrier-damage patterns of mild or moderate degree or slightly increased levels of CSF beta1-globulin were occasionally found in all 3 diseases. The results indicate that isoelectric focusing of the CSF proteins may be of diagnostic value in the cerebellar syndrome of chronic alcoholism and in essential tremor but does not reveal any characteristic abnormalities in Parkinson's disease.

Serum protein binding of lead in vitro in amyotrophic lateral sclerosis patients and controls.

The binding of lead to serum proteins was studied in vitro in ALS-patients and controls. Serum was incubated with 210Pb, the proteins were separated by isoelectric focusing and the radioactivity in the different protein fractions was determined by liquid scintillation counting. The activity was concentrated in the orosomucoid (acid alpha1-glycoprotein) fraction both in ALS-patients and controls under the present experimental conditions and the capacity of this protein to bind lead was demonstrated in a control experiment. The findings are discussed in relation to our earlier observations on increased concentrations of lead in cerebrospinal fluid and plasma in ALS-patients and the hypothetical role of the retrograde axonal transport in motoneurons in the pathogenesis of ALS. The present observations do not lend support to the idea that the increased plasma concentrations of lead in the disease would be related to qualitative differences in serum protein binding.

The physical state of the erythrocyte membrane in myotonic dystrophy.

The molecular pathology of myotonic dystrophy is believed to be expressed at the plasma membrane level. Previous assessments of membrane fluidity, a marker of the biochemical state of the membrane, have yielded conflicting results. In this study, erythrocyte membrane fluidity was reevaluated using highly sensitive fluorescence probe techniques. Steady-state anisotropy was measured with diphenylhexatriene (DPH), trimethylaminophenyl-hexatriene (TMA-DPH) and phenylhexatrienylphenylpropionic acid, probing different regions of the membrane. In the patients, significantly increased steady-state anisotropy was obtained with DPH, probing the hydrophobic core of the membrane, while slightly reduced anisotropy was found with TMA-DPH. The dynamic properties of the membrane lipids were further examined by means of time-resolved measurements with DPH. The excited state decay kinetics could best be described by a bi-exponential decay model. A large redistribution of the probe populations and a reduction of the average order parameter were found in the patients indicating a less ordered or more fluid lipid matrix. These perturbations might be induced by a protein abnormality and altered protein-lipid interaction within the erythrocyte membrane.

Effect of ethanol on synaptosomal sialic acid metabolism in the developing rat brain.

The total, glycoprotein-bound and glycolipid-bound sialic acid concentration, ad the activities of ecto-sialyltransferase and neuraminidase were determined in synaptosomes from preweanling ethanol-treated and control rats. The period of treatment corresponded to that of maximal synaptogenesis and peak synthesis of sialoglycocompounds (days 27-37 postconception). The average of the peak blood ethanol concentration was 271 mg/100 ml. In the ethanol-treated animals the sialic acid concentration was significantly reduced (approximately 20%) with an equally distributed decrease of glycoprotein- and glycolipid-bound sialic acid. The activity of ecto-sialyltransferase with asialofetuin as exogeneous acceptor was significantly diminished (about 30%) in the ethanol-treated pups. Neuraminidase showed an unchanged activity after correction for the reduction of endogeneous sialic acid substrate concentration. The total protein and lipid concentrations of the synaptosomal preparations did not differ between the groups. These results suggest that ethanol treatment during on of the vulnerable periods of brain development causes an inhibition of the incorporation of sialic acid into synaptosomal membrane-bound sialoglycocompounds. Such an effect of ethanol exposure might disturb intercellular interactions and the functional performance of the membrane during development, and could be of importance in the pathogenesis of the central nervous system manifestations of the fetal alcohol syndrome.

Glycoprotein sialyl- and galactosyl transferase activities in erythrocyte membranes in alcoholic patients and healthy controls.

In investigating possible mechanisms underlying carbohydrate deficiencies in serum transferrin and erythrocyte membranes in alcoholics, a total of 27 alcoholic patients and 27 healthy controls were examined for the activities of sialytransferase in serum and erythrocyte membranes and galactosyltransferase in erythrocyte membranes. The enzymes were assayed with endogenous and different exogenous glycoprotein acceptors and with [14C] CMP-sialic acid and [14C] UDP-galactose as substrates. No decrease in enzyme activities were found in alcoholic patients compared to controls, indicating that chronic ethanol abuse does not exert any direct inhibitory effect on these glycosyltransferases in isolated erythrocyte membranes or on sialytransferase in serum. Further studies of the effect of ethanol on the metabolism of complex carbohydrates are clearly necessary.

Reduction of the sialic acid and galactose concentrations in erythrocyte membranes in alcoholics.

The sialic acid and galactose concentrations in erythrocyte membranes were examined in 32 and 17 alcoholics respectively, during current abuse, and the sialic acid concentration in 10 patients after one and four weeks of abstention. During current alcohol abuse the concentrations of these two surface carbohydrates were decreased by an average of 10% and 16% respectively, when compared to matched, healthy controls (p less than 0.0005). After one and four weeks of abstention the sialic acid values were not significantly different from those of the controls suggesting that the abnormal cells were eliminated within one week after cessation of alcohol intake. No correlations were found between the sialic acid or galactose concentrations and peripheral red cell characteristics or serum liver enzyme levels. Hemolysis in isotonic Tris was significantly increased during current abuse, and was highly dependent on the sialic acid concentration at one particular stage of Tris-induced lysis. The mechanism behind the change of the oligosaccharide concentration of the membrane may either be increased hydrolytic cleavage or inhibition of the synthesis of glycoproteins and glycolipids. Previous similar findings in serum transferrin in alcoholics and in synaptosomes from young rat brain after ethanol exposure indicate that glycoprotein and glycolipid metabolism may be an important target, directly or indirectly, in the biological actions of ethanol.

Complex functional and structural coagulation abnormalities in the carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is an autosomal recessive disease with multisystemic manifestations. During childhood the patients may suffer from hemorrhages, which may be lethal, venous thromboses and stroke-like episodes. In this study 15 patients with CDG syndrome type I were examined from the levels and isoform patterns of coagulation factors and inhibitors and fibrinolysis parameters. The screening assays APTT and PTC were unaffected in most cases. In spite of this reduced levels were found particularly for factors II, V, X and XI and for antithrombin and protein C. Low values tended to be associated with elevated liver enzyme levels in serum. The values were at potential clinical risk levels for protein C and/or antithrombin in more than half of the patients, and for factor V and/or factor XI in one third of them. There were no current differences in values between patients who had previously displayed clinical symptoms of coagulation disturbance and those without such symptoms. Partially carbohydrate-deficient isoforms were demonstrated in antithrombin, protein C, protein S and in alpha 2-antiplasmin, but not in factors II, X and fibrinogen. Abnormal isoforms did not appear to reduce the functional activity of the respective glycoproteins. Analysis of individual hemostatic parameters is recommended in these patients in connection with clinical symptoms or elective surgery. The observed variability of the carbohydrate defect in glycoproteins in this disease may be a clue to its pathogenesis.

Denaturing high-performance liquid chromatography is a suitable method for PMM2 mutation screening in carbohydrate-deficient glycoprotein syndrome type IA patients.

The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065). The gene spans 8 exons and 741 bp of coding DNA. Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon. The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degrees C with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.

Quantitative estimation of abnormal microheterogeneity of serum transferrin in alcoholics.

A qualitative abnormality of the microheterogeneity of serum transferrin, demonstrated by isoelectric focusing, has previously been shown to be a highly specific indication of chronic alcoholism. The abnormality consists of a selective increase of a cathodal transferrin component which is probably caused by a reduction of the sialic acid content. The present study describes a method for quantitative estimation of the abnormal transferrin. The technique was based on analytical isoelectric focusing as the first step followed by direct immunofixation. The immunofixed transferrin was quantified by computerized on-line densitometry, and the transferrin abnormality was calculated as a quotient, where the amount of the cathodal component was expressed as a percentage of the relative total immunofixed transferrin quantity. This method was shown to possess high sensitivity and good reproducibility. In the controls the mean value of the quotient was 3.7%, while in the alcoholics it was 9.5% which was a highly significant difference (p less than 0.001). The possible functional significance of a disturbed sialic acid metabolism in alcoholism is discussed.