Helena Stibler

Carbohydrate-deficient glycoprotein syndromes: peculiar group of new disorders.

A new group of metabolic disorders, the carbohydrate-deficient glycoprotein (CDG) syndromes, is reviewed with emphasis on the key condition, the CDG syndrome type I. This disease, an autosomal-recessive multisystem condition, has now been diagnosed in 45 Scandinavian patients. It is characterized by carbohydrate deficiencies of a number of glycoproteins, including uniform changes in transferrin. The transferrin alterations provide a distinct biologic marker and a practical and simple laboratory diagnostic means employing analysis of serum or blood spots from Guthrie-type filter paper. The syndrome presents differently through various life periods. A four-stage grouping system by age has been constructed and is presented. During infancy, internal organ symptoms are dominant; some may be life-threatening. In later childhood and adolescence, static mental deficiency, cerebellar ataxia, slowly progressive lower limb neuropathy, and pigmentary retinal degeneration, as well as secondary skeletal deformities, are the most prominent findings. Two very recently described clinical and biologic variants, CDG syndromes II and III, are summarized and compared to CDG type I.

Biochemical Characteristics and Diagnosis of the Carbohydrate-deficient Glycoprotein Syndrome

29 patienxts with a new inherited complex developmental deficiency syndrome—the carbohydrate‐deficient glycoprotein syndrome—were studied biochemically. The most striking biochemical abnormality in these patients is the presence of secretory glycoproteins, that are deficient in their carbohydrate moieties. Serum transferrin shows the most pronounced carbohydrate defect, both quantitatively and qualitatively. Half of this glycoprotein is apparently missing two or four of its terminal trisaccharidcs—sialic acid, galactose and N‐acetylglucosamine—while the carbohydrate core appears to be intact. The abnormal transferrin is also present in the liver. This biochemical alteration is a highly specific marker of the syndrome, which can be diagnosed either qualitatively by isoelectric focusing of serum transferrin or quantitatively by the “carbohydrate‐deficient transferrin” (CDT) assay. In the CDT assay all of these patients have values approximately ten times above the reference level. The unique carbohydrate defect in secretory glycoproteins indicates that this disorder represents a new type of inborn error of glycoprotein metabolism. Studies of eleven enzymes in glycoprotein synthesis and catabolism have not revealed any deficiency of glycosidases or glycosyltransferases. The nature of the transferrin change and the cathepsin assays performed to date may suggest an as yet unidentified degradation abnormality.

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.

Early manifestations of the carbohydrate-deficient glycoprotein syndrome

We diagnosed the carbohydrate-deficient glycoprotein syndrome in five children who were seen during their first year of life with failure to thrive, feeding difficulties, psychomotor retardation, hypotonia, esotropia, inverted nipples, lipodystrophy, pericardial effusion, and hepatic dysfunction. Steatosis was observed in liver biopsy specimens, and cerebellar hypoplasia was present on computed tomography. The disorder is characterized by a complex carbohydrate deficiency in certain glycoproteins, notably transferrin, which can be used as a marker of the disease. The carbohydrate-deficient glycoprotein syndrome may be an important and easily identifiable cause of failure to thrive and neurologic dysfunction in infancy. The presence of the disorder in siblings of different gender and the finding of biochemical abnormalities in some unaffected parents suggest an autosomal recessive inheritance.

Carbohydrate-deficient transferrin (CDT) in serum in women with early alcohol addiction

Carbohydrate-deficient transferrin (CDT) in serum was determined by micro anion exchange chromatography and a transferrin radioimmune assay in 58 consecutive women treated for early alcohol dependence compared, with 62 healthy females with an alcohol consumption of 0-15 g of ethanol/day. The upper normal CDT level was 74 mg/l. CDT was elevated above this value in 83% of the alcoholic women with an intake of 60 g of ethanol/day or more for at least 7 days within the preceding two weeks. CDT values were significantly positively correlated with daily alcohol consumption but not with GT, ASAT, ALAT or MCV. During abstinence CDT level declined exponentially with a half-life of 14 +/- 3 days. The results indicated that CDT may be as sensitive and specific a marker in women with early alcohol addiction as in previously studied male alcoholics. The amount of alcohol consumed appeared to be more important than sex or liver function. Determination of CDT may thus offer a means for early objective diagnosis and adequate treatment also of women in early stages of alcoholism.

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.

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 multi‐systemic 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 μM (range 22‐42 μM) to a trough value of 72 μM (range 39–103 μM). No serious side effects were observed. Surprisingly, the mean serum concentration of four glycoproteins (transferrin, α1‐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.

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.

Diagnosis of the carbohydrate‐deficient glycoprotein syndrome by analysis of transferrin in filter paper blood spots

Carbohydrate‐deficient glycoprotein syndrome is a recently identified recessively inherited, multisystemic disease with severe nervous system involvement. It is characterized biochemically by carbohydrate‐deficient serum glycoproteins, and can be diagnosed by analysis of abnormal isoforms of serum transferrin. Using stored, neonatally collected filter paper blood spots from such patients, it was shown that neonatal diagnosis was possible by immune‐isoelectric focusing of transferrin eluted from up to 14‐year‐old samples. Freshly collected blood on filter paper was readily analyzed quantitatively for carbohydrate‐deficient isotransferrins by a rapid microchromatographic assay, revealing highly elevated values in all patients. The presently described methods thus provide a means for early diagnosis of the carbohydrate‐deficient glycoprotein syndrome in microliter volumes of capillary blood. Sampling on filter paper offers an important simplification in sample collection, storage and transport, and may make population studies possible.

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.