Changes in diaphragm structure following prolonged mechanical ventilation in piglets.

BACKGROUND: Prolonged mechanical ventilation and inactivity negatively affect muscle function. The mechanisms for this dysfunction are unclear and clinical studies of respiratory muscle are difficult to carry out. An animal model simulating the critical care environment was used to investigate the effects of 5 days' mechanical ventilation and diaphragm inactivity on diaphragm muscle morphology. METHODS: Twelve 2-4-month-old piglets weighing 23-30 kg were studied. Seven animals received controlled mechanical ventilation and sedation such that spontaneous breathing efforts were inhibited over 5 days. Five control animals were ventilated for only 4-6 h following surgical preparation. Diaphragm biopsies were obtained from the left costal region at the end of all experiments. RESULTS: Morphometric, morphologic, electron microscopic and enzyme histochemical examination of costal diaphragm biopsies was carried out. Contractile properties were studied over 5 days and the results have been previously reported. Cross-sectional area of alI fiber types was increased compared with controls. The proportion of type IIb/x fibers increased following inactivity (P < 0,05) and the proportion of type I and IIa fibers tended to decrease although not significantly. Focal areas of diaphragm fiber regeneration were found without signs of inflammation. Increased appearance of cytoplasmic vacuoles consisting of lipid accumulation was noted in type I fibers. Several study animals developed focal areas with weak myofibrillar ATPase activity and disrupted fiber organization. There were areas of myofibrillary destruction and loss of sarcomeric pattern, without evidence of selective thick filament loss or a change in the myosin to actin ratio. CONCLUSION: Five days' mechanical ventilation with sedation and complete diaphragm inactivity resulted in changes in muscle fiber structure. A causal relationship can not be concluded but the acute changes in fiber type distribution and structure suggest that previously reported diaphragm contractile impairment occurs at the level of muscle fibers.

Scandinavian CDG-Ia patients: genotype/phenotype correlation and geographic origin of founder mutations.

Congenital disorders of glycosylation type Ia, (previous name carbohydrate-deficient glycoprotein syndrome type Ia; CDG-Ia) is an inherited disorder of the glycosylation of certain glycoproteins. The defect is caused by mutations in the phosphomannomutase 2 (PMM2) gene located in chromosome region 16p13. The purpose of this study was twofold: (1) to investigate the possible correlation between certain genotypes and the phenotype of the patients and their PMM activity, and (2) to study further the founder origin of the Scandinavian mutations. Sixty-four CDG-Ia patients were studied. Regardless of mutation combination, the patients showed the basic neurological symptoms associated with CDG-Ia. However, patients carrying the mutation 548T-->C had less severe disease, e.g., no pericardial effusions, malnutrition, or clinical coagulation disturbances. Liver dysfunction and peripheral neuropathy were milder. In contrast, patients carrying mutation 691G-->A showed a high incidence of severe malnutrition and hepatopathy, and they had the highest mortality including affected siblings. Heterozygotes for the two most common mutations (422G-->A and 357C-->A) displayed a phenotype of variable severity sometimes leading to early death. PMM activity showed no correlation with either genotype or phenotype but was reduced in most patients. There was a pronounced geographic clustering for some of the Scandinavian mutations. For example, 548T-->C was almost exclusively found in patients stemming from southeastern parts of Sweden, whereas 26G-->A was found to cluster in a region in the most southern parts of Sweden, suggesting that these mutations originated in these two regions separately as founder mutations. The most frequent mutation (422G-->A) did not show a specific geographic focus. The widespread 422G-->A mutation is probably an older mutation, although haplotype data from intragenic polymorphisms indicate that this mutation also arose only once. The detailed information of the origin of mutations and their respective associated phenotypic pattern should enable improvements to be made regarding tools for genetic counseling and for prenatal diagnoses in CDG-Ia families.

Interference of transferrin isoform types with carbohydrate-deficient transferrin quantification in the identification of alcohol abuse.

BACKGROUND: Isoforms of transferrin interfere with measurement of carbohydrate-deficient transferrin (CDT) as a marker of heavy alcohol consumption. We evaluated the rate of inaccurate CDT results by immunoassays. METHODS: We studied 2360 consecutive sera (1614 individuals) submitted for CDT assay without clinical information as well as samples from 1 patient with a congenital disorder of glycosylation (CDG Ia) and from 6 healthy carriers of CDG Ia. The CDTect, %CDT-TIA, and new %CDT immunoassays were compared with HPLC (%CDT-HPLC). Transferrin isoform pattern were evaluated by isoelectric focusing (IEF). RESULTS: Transferrin BC and CD heterozygotes were found at frequencies of approximately 0.7% and approximately 0.2%, respectively. Another transferrin C subtype, where di- and trisialotransferrin partly coeluted (tentatively identified as C2C3), was observed in approximately 0.6%. Compared with the %CDT-HPLC method, the immunoassays often produced low results for transferrin BC and high results for transferrin CD and "C2C3". A very high trisialotransferrin value (frequency approximately 1%) often produced high CDT immunoassay results. In four of six healthy carriers of CDG Ia, a- and disialotransferrin were highly increased and the HPLC and IEF isoform patterns were indistinguishable from those in alcohol abuse. CONCLUSIONS: Rare transferrin isoform types and abnormal amounts of trisialotransferrin (total frequency approximately 2-3%) may cause incorrect determination of CDT with immunoassays. The observed variants were readily identified by HPLC and IEF, which can be recommended for verification of CDT immunoassay results in doubtful cases. In healthy carriers of CDG Ia, CDT is high by all assays.

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.

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.

A neurodystrophic syndrome resembling carbohydrate-deficient glycoprotein syndrome type III.

A 10-month old girl is described with a serum transferrin isoform abnormality of the same kind as in two previously reported girls with carbohydrate-deficient glycoprotein syndrome type III. This patient presented with joint abnormalities and rapidly developing hypsarrhythmia, hypotonia, psychomotor delay and growth retardation. Fingers, toes, nails and local skin were dysmorphic. She had pale optic discs, thoracic syringomyelia and frontal lobe atrophy at three months. The CDT value in serum was greatly elevated. Several carbohydrate-deficient isoforms were found in transferrin (four), alpha1-antitrypsin (three), antithrombin (two) and thyroxine-binding globulin (four). Mutations in the CDGS 1-gene were excluded. The CDGS III glycoprotein abnormality most probably represents a distinct disorder of glycoprotein metabolism, and needs to be considered in unclear hypsarrhythmia with developmental delay. Dysmorphic features may be added to this syndrome.

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.

Failure of short-term mannose therapy of patients with carbohydrate-deficient glycoprotein syndrome type 1A.

Carbohydrate-deficient glycoprotein syndrome type 1A (CDGS1A) is an inherited disorder with multisystemic abnormalities resulting from failure to generate sufficient lipid-linked oligosaccharide precursor or to transfer the sugar chain to many glycoproteins. Cultured fibroblasts from these patients have reduced incorporation of mannose into glycoproteins which can be corrected by adding D-mannose to the culture medium. Providing dietary mannose to elevate mannose concentrations in vivo therefore might remedy some of the underglycosylation in the patients. Five children with CDGS1A aged 15 months to 14 y completed a protocol of enteral supplementation with D-mannose 100 mg/kg every 3 h for 9 d. The mean S-mannose level increased from 32 microM (range 22-42 microM) to a trough value of 72 microM (range 39-103 microM). No serious side effects were observed. Surprisingly, the mean serum concentration of four glycoproteins (transferrin, alpha1-antitrypsin, antithrombin, and thyroxine-binding globulin) tended to decrease, and the mean serum concentration of carbohydrate-deficient transferrin (CDT) increased. Furthermore, the initially present abnormal isoforms of these glycoproteins and of protein C became more prominent and/or additional abnormal isoforms appeared. This short-term trial does not support a benefit of mannose to the deficient glycosylation of CDGS1A patients.

Intestinal, pancreatic and hepatic involvement in carbohydrate-deficient glycoprotein syndrome type I.

BACKGROUND: Children with carbohydrate-deficient glycoprotein syndrome type I during infancy have gastrointestinal symptoms and growth impairment, the cause of which is largely unknown. METHODS: Seven children were investigated with small intestinal biopsy, liver biopsy, duodenal intubation with determination of lipolytic and proteolytic activity, and test meal. Weight, length-height, and head circumference were recorded regularly. RESULTS: Growth was affected from early infancy, with an initial low rate of weight gain followed by impaired linear growth. Vomiting and diarrhea were dominant symptoms. Four of seven children had abnormal findings in light microscopic examination of small intestinal biopsy specimens, with short villi and increased inflammatory cells in the stroma, that did not respond to elimination of such food proteins as cow's milk or gluten. Electron microscopic study showed dilatation of smooth endoplasmic reticulum and abnormal inclusions containing lipids. The liver was abnormal in all. Besides steatosis and fibrosis or cirrhosis, there was a remarkable increase of inflammatory cells in portal zones. Activity of lipolytic enzymes in duodenal juice was low, except in one child, who no longer had growth problems or symptoms. Two of six had abnormal proteinolytic activity in duodenal juice. Digestion of triglycerides and absorption were within normal limits, as was the absorption of glucose and xylose. CONCLUSIONS: Inflammation of small intestine and liver may be the cause of gastrointestinal symptoms. In all likelihood, the growth failure was because of low caloric intake and increased losses related to vomiting. Growth and gastrointestinal symptoms improved spontaneously as time elapsed.

The heart and pericardial effusions in CDGS-I (carbohydrate-deficient glycoprotein syndrome type I).

Pericardial effusions were found in 6 of 10 children with carbohydrate-deficient glycoprotein syndrome type I (CDGS-I). In three cases pericardectomy was necessary. Blood concentrations of several glycoproteins and albumin were low. Similar abnormal isoforms of four glycoproteins were found in blood (B) and pericardial fluid (PF). There was a significant negative correlation between the mean concentration ratio PF/B and the molecular mass (MW) of 11 proteins. For proteins with MW < 100 kDa there were significant correlations in the controls, but not in the patients, between the PF/B ratio and both the MW and the sialic acid contents in the (glyco-)proteins. The pericardium exhibited focal mixed inflammatory changes with mesothelial proliferation, with widened endoplasmic reticulum and flocculent and/or lamellated material. Damage to a pericardial protein barrier is suggested to be involved in pericardial effusion in CDGS-I.

Isoforms and levels of transferrin, antithrombin, alpha(1)-antitrypsin and thyroxine-binding globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein syndrome type I (CDGS I) is an autosomal recessive disease with multiple organ manifestations. The diagnostic biochemical marker has been typical carbohydrate-deficient isoforms of transferrin (Tf). Many other glycoproteins in blood may show similar defects, but have not been systematically studied before. Forty-eight CDGS I patients and 22 controls were examined for total concentrations and isoform distribution of Tf, antithrombin (AT), alpha(1)-antitrypsin (alpha(1)-AT) and thyroxine-binding globulin (TBG), and for the level of carbohydrate-deficient transferrin (CDT). The absolute values varied with age. The most frequent persistent quantitative changes were reduced levels of AT (97%) and elevated CDT values (100%). Isoforms lacking one to eight of four to eight possible sialic acid residues were found in AT, TBG and Tf in all cases, with variable intensity and frequency, and in all except one patient in alpha(1)-AT. The isoform changes were most constant and pronounced in Tf. The other three glycoproteins showed more abnormal heterogeneity in the youngest than in the older patients. The results indicated that the biochemical defect stabilizes with age, and suggested partial hypoglycosylation rather than non-glycosylation of these glycoproteins. Analysis of Tf isoforms is still the safest diagnostic marker of CDGS I from full-term birth and over the ages.

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.

[CDGS-1--a recently discovered hereditary metabolic disease. Multiple organ manifestations, incidence 1/80,000, difficult to treat]. / CDGS-1--en nyupptäckt ärftlig metabolisk sjukdom. Manifestationer från många organ, incidens 1/80,000, svårbehandlad.

Carbohydrate-deficient glycoprotein syndrome type 1 (CDGS-1) is an autosomal recessive hereditary metabolic disorder, the gene locus of which is chromosome 16p13. The disorder is characterised by genetic heterogeneity, and by decrease in the gene product, phosphomannomutase 2, though the heterogeneity is far less manifest in affected Swedish families. Its incidence is 1/80,000 live births, and the under-5 mortality rate over 30 per cent. The causes of death are liver failure, cardiac tamponade, haemorrhaging, and severe infection. The characteristic biochemical aberration is the occurrence of deficient carbohydrate chains in many but not all circulating glycoproteins, and the serum and blood concentrations of some glycoproteins may be above or below normal. These changes may improve over time, but never normalise. The clinical picture is generally more problematic during the first years of life when psychomotor retardation is complicated by failure to thrive, liver dysfunction, pericardial effusions, and stroke-like episodes. In addition, strabismus, lipocutaneous anomalies, and gluteal fat pads are always present, and muscular hypotonia and restricted joint mobility are common. Failure to thrive is common, with vomiting and diarrhoea and subsequent slow growth. Inflammation is a constant finding in the liver, and very common in the small bowel. Pancreatic function is also affected. Pericardial effusion has been reported in 50 per cent of the youngest children, requiring pericardectomy in 30 per cent of cases. Haemorrhaging and thromboembolic complications may occur, and the serum concentrations of several factors and inhibitors are low, particularly those of factors V and XI, protein C and antithrombin. Stroke-like episodes occur in about 30 per cent of cases, often following an infection, with coma lasting for hours to several days. Such sequelae as hemiplegia, blindness, and other focal neurological pathology have been observed transiently. Diagnosis is based on the serum carbohydrate-deficient transferrin level, verified by isoelectric focusing. Molecular genetic procedures enable point mutations to be identified and prenatal diagnosis to be performed in many families.

Immunoglobulin levels in patients with carbohydrate-deficient glycoprotein syndrome type I.

BACKGROUND: The characteristic feature of carbohydrate-deficient glycoprotein syndrome (CDGS) type I, a multisystemic disease, is underglycosylation of many serum glycoproteins, such as transferrin. A few cases of severe infections during childhood have been reported and an underlying immunodeficiency has been suggested. Because of this and the fact that all immunoglobulin (Ig) isotypes are glycoproteins we analysed the Ig levels in patients with CDGS I. METHODS: The serum concentrations of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE, and the frequency of the G2m(23) allotype were measured by enzyme immunoassay in 15 patients with CDGS type I. RESULTS: Ten (67%) patients had an elevated level of at least one Ig, when compared to age-related reference ranges. No particular isotype was involved although a tendency towards high IgE levels was registered. The frequency of homozygous G2m(23)-negative CDGS patients (33%) was not different from that of blood donors (34%). CONCLUSION: We conclude that CDGS I patients have no major changes in the serum levels of any specific Ig isotype. The severe infections observed in some CDGS patients are therefore unlikely to involve any Ig deficiency. Our results do not exclude that Ig of patients with CDGS may have altered physiological functions because of abnormal glycosylation.

Fine mapping of the gene for carbohydrate-deficient glycoprotein syndrome, type I (CDG1): linkage disequilibrium and founder effect in Scandinavian families.

Carbohydrate-deficient glycoprotein syndrome type I (CDG I) is characterized clinically by severe nervous system involvement and biochemically by defects in the carbohydrate residues in a number of serum glycoproteins. The CDG1 gene was recently localized by us to a 13-cM interval in chromosome region 16p13. In this study 44 CDG I families from nine countries were analyzed with available markers in a region ranging from marker D16S495 to D16S497, and haplotype and linkage disequilibrium analyses were performed. One specific haplotype was found to be markedly overrepresented in CDG I patients from a geographically distinct region in Scandinavia, strongly indicating that CDG I families in this region share the same ancestral CDG1 mutation. furthermore, analysis of the extent of the common haplotype in these families indicates that the CDG1 gene is located in the region defined by markers D16S513-AFMa284wd5-D16S768-D16S406-D16S502 . The critical CDG1 region, in strong linkage disequilibrium with markers AFMa284wd5, D16S768, and D16S406, thus constitutes less than 1 Mb of DNA and less than 1 cM in the very distal part of the CDG1 region defined by us previously.

Carbohydrate-deficient transferrin in galactosaemia.

Carbohydrate-deficient isoforms of transferrin (CDT) were examined in Guthrie cards from patients with galactosaemia before and during dietary treatment for up to 9 y. In untreated patients the CDT values were elevated due to abnormal asialo- and/or disialotransferrin. During treatment, the CDT levels were normal except on a few temporary occasions. Galactose or its close metabolites did not inhibit two relevant glycosyltransferases in vitro, and their levels were not correlated to the CDT values. The transferrin isoform changes in untreated patients were similar to, but less pronounced than in CDG syndrome type I.

[Jaeken's (CDG) syndrome in two sisters]. / Zespól Jaekena (CDG) u dwóch sióstr.

Jaeken's syndrome or the carbohydrate-deficient glycoprotein (CDG) syndrome, is a newly recognized metabolic syndrome with poor weight gain in children, and multisystematic abnormalities, mainly due to defective carbohydrate entities in many glycoproteins, leading to neurologic dysfunction. Using the standardized method of phenotype evaluation with computer assistance according to the Munich Dysmorphologic Database, two sisters with CDGs were examined to decide if this metabolic entity contains dysmorphic features characterising dysmorphic syndromes. Diagnosis was based on clinical symptomatology and transferrin isoforms which showed tetrasialotransferrin deficiency and increased disialotransferrin in serum. Dysmorphic studies can be helpful in recognition of this syndrome, now described for the first time in Poland.

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.

Normal pubertal development in a female with carbohydrate deficient glycoprotein syndrome.

A girl is reported who presented with many of the clinical and biochemical characteristics of type I carbohydrate deficient glycoprotein syndrome. Unusually, however, she experienced a normal pubertal development.