Alfried Kohlschütter

Friedreich's ataxia : Point mutations and clinical presentation of compound heterozygotes

Friedreichs ataxia is the most common inherited ataxia. Ninety‐six percent of patients are homozygous for GAA trinucleotide repeat expansions in the first intron of the frataxin gene. The remaining cases are compound heterozygotes for a GAA expansion and a frataxin point mutation. We report here the identification of 10 novel frataxin point mutations, and the detection of a previously described mutation (G130V) in two additional families. Most truncating mutations were in exon 1. All missense mutations were in the last three exons coding for the mature frataxin protein. The clinical features of 25 patients with identified frataxin point mutations were compared with those of 196 patients homozygous for the GAA expansion. A similar phenotype resulted from truncating mutations and from missense mutations in the carboxy‐terminal half of mature frataxin, suggesting that they cause a comparable loss of function. In contrast, the only two missense mutations located in the amino‐terminal half of mature frataxin (D122Y and G130V) cause an atypical and milder clinical presentation (early‐onset spastic gait with slow disease progression, absence of dysarthria, retained or brisk tendon reflexes, and mild or no cerebellar ataxia), suggesting that they only partially affect frataxin function. The incidence of optic disk pallor was higher in compound heterozygotes than in expansion homozygotes, which might correlate with a very low residual level of normal frataxin produced from the expanded allele. Ann Neurol 1999;45:200–206

Cumulative incidence rates of the mucopolysaccharidoses in Germany.

SummaryIn order to estimate the cumulative incidence rates of the mucopolysaccharidoses (MPS) in Germany, a retrospective epidemiological survey covering the period between 1980 and 1995 was implemented. Multiple ascertainment sources were used to identify affected patients. A prevalence of approximately 0.69 cases per 100 000 births was obtained for MPS I (Hurler phenotype). Within the study period, 4 patients with Hurler/Scheie phenotype and 7 cases with Scheie disease were detected. The cumulative incidence for MPS II (Hunter syndrome) was estimated as 0.64 cases per 100 000 births (1.3 cases per 100 000 male live births); that for MPS III (Sanfilippo syndrome types A, B and C) as 1.57 cases in 100 000 births; that for MPS IV A (Morquio syndrome) as 0.38 cases in 100 000; and that for MPS VI (Maroteaux–Lamy syndrome) as 0.23 cases per 100 000 births. Two cases of MPS IVB (β-galactosidase deficiency) have been identified, but no patients with MPS VII or MPS IX. A relatively high number of patients with MPS IIIB, MPS IVA and MPS VI were of Turkish origin. The crude rate for all types of mucopolysaccharidoses is approximately 3.53 cases in 100 000 live births. The cumulative incidence pattern of MPS in Germany was compared with the corresponding rates among other industrial nations obtained from recent literature: the crude cumulative rates for all types of mucopolysaccharidoses (3.4–4.5 in 100 000 live births) were similar among all published populations; however, different frequencies of the various forms of MPS were observed.

Beneficial effects of α-lipoic acid and ascorbic acid on endothelium-dependent, nitric oxide-mediated vasodilation in diabetic patients: relation to parameters of oxidative stress

The impairment of nitric oxide (NO)-mediated vasodilation in diabetes has been attributed to increased vascular oxidative stress. Lipoic acid has been shown to have substantial antioxidative properties. The aim of this study was to assess the effect of lipoic acid on NO-mediated vasodilation in diabetic patients in comparison with the well-recognized effect of ascorbic acid. Using venous occlusion plethysmography, we examined the effects of lipoic acid (0.2 mM) and ascorbic acid (1 and 10 mM) on forearm blood flow responses to acetylcholine, sodium nitroprusside and concomitant infusion of the NO-inhibitor, N(G)-monomethyl-L-arginine, in 39 diabetic patients and 11 control subjects. Plasma levels of antioxidants and parameters of lipid peroxidation were measured and correlated to endothelial function tests. Lipoic acid improved NO-mediated vasodilation in diabetic patients, but not in controls. NO-mediated vasodilation was improved by ascorbic acid at 10 mM, but not 1 mM. Improvements of endothelial function by ascorbic acid and lipoic acid were closely related. The beneficial effects of lipoic acid were positively related to plasma levels of malondialdehyde and inversely related to levels of ubiquinol-10. These findings support the concept that oxidative stress contributes to endothelial dysfunction and suggest a therapeutic potential of lipoic acid particularly in patients with imbalance between increased oxidative stress and depleted antioxidant defense.

ALPHA -TOCOPHEROL AS A REDUCTANT FOR CU(II) IN HUMAN LIPOPROTEINS : TRIGGERING ROLE IN THE INITIATION OF LIPOPROTEIN OXIDATION

Initiation of lipid peroxidation by Cu(II) requires reduction of Cu(II) to Cu(I) as a first step. It is unclear, however, whether this reaction occurs in the course of lipoprotein oxidation. It is also unknown which reductant, if any, can drive the reduction of Cu(II) in this case. We found that Cu(II) was rapidly reduced to Cu(I) by all major human lipoproteins (high, low, and very low density lipoproteins (HDL, LDL, and VLDL), and chylomicrons). Cu(II)-reducing activity was associated with a lipid moiety of the lipoproteins. The rates of Cu(II) reduction by different lipoproteins were similar when the lipoproteins were adjusted to similar α-tocopherol concentrations. Enriching lipoproteins with α-tocopherol considerably increased the rate of Cu(II) reduction. Cu(II) reduction by α-tocopherol-deficient LDL isolated from a patient with familial inherited vitamin E deficiency was found to occur much slower in comparison with LDL isolated from a donor with a normal plasma level of α-tocopherol. Initial rate of Cu(II) reduction by α-tocopherol-deficient LDL was found to be zero. Enriching LDL with ubiquinol-10 to concentrations close to those of α-tocopherol did not influence the reaction rate. When LDL was treated with ebselen to eliminate preformed lipid hydroperoxides, the reaction rate was also not changed significantly. Cu(II) reduction was accompanied by a consumption of lipoprotein α-tocopherol and accumulation of conjugated dienes in the samples. Increasing α-tocopherol content in lipoproteins slightly decreased the rate of conjugated diene accumulation in LDL and HDL and considerably increased it in VLDL. The results suggest that α-tocopherol plays a triggering role in the lipoprotein oxidation by Cu(II), providing its initial step as follows: αTocH + Cu(II) αToc + Cu(I) + H. This reaction appears to diminish or totally eliminate the antioxidative activity of α-tocopherol in the course of lipoprotein oxidation.

Plasma ubiquinol-10 is decreased in patients with hyperlipidaemia

Ubiquinol-10, the reduced form of ubiquinone-10 (coenzyme Q10), is a potent lipophilic antioxidant present in nearly all human tissues. The exceptional oxidative lability of ubiquinol-10 implies that it may represent a sensitive index of oxidative stress. The present study was undertaken to assess the hypothesis that the level of ubiquinol-10 in human plasma can discriminate between healthy subjects and patients who are expected to be subjected to an increased oxidative stress in vivo. Using a newly developed method, we measured plasma ubiquinol-10 in 38 hyperlipidaemic patients with and without further complications, such as coronary heart disease, hypertension, or liver disease, and in 30 healthy subjects. The oxidizability of plasma samples obtained from hyperlipidaemic patients was found to be increased in comparison with control subjects, suggesting that the patients were subjected to a higher oxidative stress in vivo than the controls. Plasma ubiquinol-10, expressed as a percentage of total ubiquinol-10 + ubiquinone-10 or normalized to plasma lipids, was lower in the patients than in controls (P = 0.001 and 0.008, respectively). The proportion of ubiquinol-10 decreased in the order young controls > aged controls > hyperlipidaemic patients without complications > hyperlipidaemic patients with complications (P = 0.003). A negative correlation was found between the proportion of ubiquinol-10 and plasma triglycerides. The hyperlipidaemic patients with hypertension had a lower proportion of ubiquinol-10 than subjects without. When the study population was divided into smokers and non-smokers, plasma ubiquinol-10 was found to be reduced amongst smokers, independently of whether it was expressed as a percentage of total ubiquinol-10 + ubiquinone-10 (P = 0.006) or normalized to plasma lipids (P = 0.009). These data suggest that the level of ubiquinol-10 in human plasma may represent a sensitive index of oxidative stress in vivo especially indicative of early oxidative damage. Measuring plasma ubiquinol-10 can be proposed as a practical approach to assess oxidative stress in humans.

Impaired plasma antioxidative defense and increased nontransferrin-bound iron during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation

To analyze the effects of radiochemotherapy on the pro-oxidative/antioxidative balance in plasma, we measured the total radical antioxidant parameter of plasma (TRAP) and single plasma antioxidants (uric acid, sulfhydryl groups, alpha-tocopherol, ubiquinone-10/total coenzyme-Q10 ratio, ascorbate, and bilirubin) every 12 h during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation (BMT). Nontransferrin-bound iron (NTBI) was monitored as a potential pro-oxidant. Plasma levels of polyunsaturated fatty acids (PUFA) were measured as substrates, and thiobarbituric acid-reactive substances (TBARS) were measured as products of lipid peroxidation. Allantoin was analyzed as the product of uric acid oxidation. Patients receiving busulfan, VP-16, and cyclophosphamide (BU/VP/CY) (n = 8) were compared with those receiving total body irradiation in addition to VP-16 and cyclophosphamide (TBI/VP/CY) (n = 8). TRAP values were within the normal range before therapy and decreased after BU/VP/CY by 37% (p <. 02) and after TBI/VP/CY by 39% (p <.02). During TBI and after VP-16, a temporary increase in TRAP values occurred, which was not related to changes in individual antioxidants. In vitro experiments confirmed that VP-16 had an antioxidative effect. The concentration of uric acid declined in both groups and correlated with TRAP (BU/VP/CY: r =.80, p <.001; TBI/VP/CY: r =.84, p <.001). Levels of NTBI, which is normally not found in plasma, increased rapidly during conditioning therapy (p <.02 in both groups) and correlated inversely with TRAP (weighted intraindividual Spearman rank correlation coefficient for both groups: NTBI and TRAP: r = -.59, p <.001) and PUFA (in the radiochemotherapy group: r = -.67, p <.001). Whereas PUFA declined (p <.02 in both groups), TBARS increased (p <. 05 in both groups). Furthermore, an increase of allantoin and ubiquinone-10/total coenzyme-Q10 ratio in the BU/VP/CY group was found (allantoin: p <.02; ubiquinone-10/total coenzyme-Q10 ratio: p <.05). Antioxidants only partially recovered to baseline values until day 14 after BMT. Our findings indicate oxidative stress after high-dose radiochemotherapy and suggest a contribution of NTBI therein.

Leukodystrophy incidence in Germany

Through a survey of all departments of pediatrics, neurology and neuropathology in Germany, we calculated the incidence of all major forms of leukodystrophy. Only diagnoses based on specific biochemical tests in association with typical findings and/or neuroradiologically proven white matter involvement were accepted. In accordance with these strict criteria, 617 cases of leukodystrophy were found (incidence of all forms: app. 2.0/100,000). Minimal incidence was estimated at 0.8/100,000 for adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN), 0.6/100,000 for metachromatic leukodystrophy (MLD), and 0.6/100,000 for Krabbe disease. Thus ALD/AMN is apparently underdiagnosed in Germany. A considerable proportion of leukodystrophies could not be classified in spite of adequate diagnostic procedures in experienced centers.

RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection.

Congenital cytomegalovirus brain infection without symptoms at birth can cause a static encephalopathy with characteristic patterns of brain abnormalities. Here we show that loss-of-function mutations in the gene encoding the RNASET2 glycoprotein lead to cystic leukoencephalopathy, an autosomal recessive disorder with an indistinguishable clinical and neuroradiological phenotype. Congenital cytomegalovirus infection and RNASET2 deficiency may both interfere with brain development and myelination through angiogenesis or RNA metabolism.

Defective Endoplasmic Reticulum-resident Membrane Protein CLN6 Affects Lysosomal Degradation of Endocytosed Arylsulfatase A

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a ∼27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.

NCL diseases — clinical perspectives☆

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage disorders and together are the most common degenerative brain diseases in childhood. They are a group of disorders linked by the characteristic accumulation of abnormal storage material in neurons and other cell types, and a degenerative disease course. All NCLs are characterized by a combination of dementia, epilepsy, and motor decline. For most childhood NCLs, a progressive visual failure is also a core feature. The characteristics of these symptoms can vary and the age at disease onset ranges from birth to young adulthood. Genetic heterogeneity, with fourteen identified NCL genes and wide phenotypic variability render diagnosis difficult. A new NCL classification system based on the affected gene and the age at disease onset allows a precise and practical delineation of an individual patients NCL type. A diagnostic algorithm to identify each NCL form is presented here. Precise NCL diagnosis is essential not only for genetic counseling, but also for the optimal delivery of care and information sharing with the family and other caregivers. These aspects are challenging because there are also potential long term complications which are specific to NCL type. Therefore care supported by a specifically experienced team of clinicians is recommended. As the underlying pathophysiological mechanism is still unclear for all NCL forms, the development of curative therapies remains difficult. This article is part of a Special Issue entitled: The neuronal ceroid lipofuscinoses or Batten Disease.