Volumetric Description of Brain Atrophy in Neuronal Ceroid Lipofuscinosis 2: Supratentorial Gray Matter Shows Uniform Disease Progression.

BACKGROUND AND PURPOSE: Experimental therapies for ceroid lipofuscinosis, neuronal, 2 (CLN2), a genetic disorder of childhood associated with progressive brain atrophy, are currently being developed. Because quantitative descriptions of the natural course of brain volume loss are needed to evaluate novel therapies, we performed MR imaging volumetry of patients with CLN2 to identify a suitable MR imaging marker of disease progression. MATERIALS AND METHODS: Thirteen patients (8 females, 5 males) were recruited from a prospective natural disease cohort of patients with neuronal ceroid lipofuscinosis. Repeated MR imaging volumetric analysis (29 datasets) was performed by using the FreeSurfer Software Suite. Follow-up time ranged from 8 months to 5.3 years. MR imaging-segmented brain volumes were correlated to patient age and clinical scores. RESULTS: Segmented brain volumes correlated significantly with patient age (lateral ventricles, r = 0.606, P = .001; supratentorial cortical GM, r = -0.913, P < .001; supratentorial WM, r = -0.865, P < .001; basal ganglia/thalamus, r = -0.832, P < .001; cerebellar GM, r = -0.659, P < .001; cerebellar WM, r = -0.830, P < .001) and clinical scores (lateral ventricles, r = -0.692, P < .001; supratentorial cortical GM, r = 0.862, P < .001; supratentorial WM, r = 0.735, P < .001; basal ganglia/thalamus, r = 0.758, P < .001; cerebellar GM, r = 0.609, P = .001; cerebellar WM, r = 0.638, P < .001). Notably, supratentorial cortical GM showed a uniform decline across the patient cohort. During late stages of the disease when the clinical score was zero, segmented brain volumes still correlated with patient age; this finding suggests that MR imaging volumetry allows quantitative assessment of disease progression at stages when it cannot be detected by clinical assessment alone. CONCLUSIONS: Automated MR imaging volumetry, as a nonsubjective and highly sensitive tool, is feasible in CLN2 disease and provides a quantitative basis to evaluate novel experimental therapies.

Novel morphological macular findings in juvenile CLN3 disease.

AIMS: Juvenile CLN3 disease, one of the most common forms of a group of lysosomal storage diseases called neuronal ceroid lipofuscinoses (NCLs), is a progressive neurodegenerative disorder with initial visual deterioration. The objective of this study was to analyse the retinal phenotype of patients with CLN3 disease with the help of recent ophthalmic imaging modalities to distinguish CLN3 disease from other inherited retinal dystrophies. METHODS: Patients underwent ophthalmic evaluations, including anterior and posterior segment examinations, optical coherence tomography, fundus autofluorescence, near infrared imaging and fundus photography. Patients were also assessed according to the Hamburg juvenile NCL (JNCL) score. Each ophthalmic finding was assessed by three independent examiners and assigned to a clinical severity score. RESULTS: 22 eyes of 11 patients were included. The mean age at examination was 14.4 years (range 11.8-26.4 years), with an average age at initial diagnosis of 8 years (range 4.5-11 years). The mean Hamburg JNCL score was 7.3 (range 0-13). All patients showed a specific macular striation pattern on optical coherence tomography that was independent of age and progression of the disease. Other previously described retinal features of CLN3 disease were classified into four severity grades. CONCLUSIONS: This study represents the first prospective observational case series documenting retinal abnormalities in CLN3 disease with the aid of the spectral domain optical coherence tomography. The major finding was a characteristic, striated macular pattern in all patients studied. Particularly in early disease cases, macular striae can potentially help to discriminate CLN3 disease from other inherited forms of retinitis pigmentosa.

Protracted course of juvenile ceroid lipofuscinosis associated with a novel CLN3 mutation (p.Y199X).

The juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease, MIM 204200), is an autosomal recessive lysosomal storage disease, which is characterized by ubiquitous accumulation of the lipopigment material ceroid-lipofuscin. It manifests with loss of vision in childhood due to retinal degeneration, followed by seizures and parkinsonism leading to premature death at around 30 years. Eighty-five percent of JNCL patients carry a disease-causing 1.02 kb deletion in the CLN3 gene on chromosome 16. Here we report on a large consanguineous Lebanese family with five affected siblings. Electron microscopy of lymphocytes revealed the presence of fingerprint profiles suggesting JNCL. However, disease progression, especially of mental and motor function was slower as expected for 'classic' JNCL. We thus confirmed the diagnosis by genetic testing and found a new c.597C>A transversion in exon 8, homozygous in all affected family members and not present in 200 alleles of normal controls. The mutation generates a premature termination codon (p.Y199X) truncating the CLN3 protein by 55%. In heterozygous state mutant mRNA transcripts are expressed at the same levels as the wild-type ones, suggesting the absence of nonsense mediated messenger decay. We discuss a potential residual catalytic function of the truncated protein as a cause for the mild phenotype.

[Leukodystrophies: diseases of white matter of the nervous system]. / Netzwerkprojekte für die Erforschung von Leukodystrophien, einer Gruppe seltener Erkrankungen der weissen Substanz des Nervensystems.

Leukodystrophies are a group of rare, genetic diseases, which affect myelin, the major constituent of brain and spinal cord white matter. Patients suffer from numerous, progressive neurologic symptoms, which frequently cause death. The molecular defects causing leukodystrophies are heterogeneous. For some leukodystrophies the genetic cause is known, whereas for others the disease-causing gene has not yet been identified. Except for a few leukodystrophies, in which bone marrow transplantation is an option to prevent disease progression, curative therapy is not available. However, clinical trials involving gene therapy and enzyme replacement therapy have been initiated as a result of accomplishments in basic research. The development of magnetic resonance imaging and spectroscopy has improved the diagnosis of leukodystrophies. Nevertheless, due to the insidious and frequently non-characteristic onset of leukodystrophies and the fact that most primary physicians have never before encountered patients with these rare diseases, diagnosis is often delayed. In Germany, the Federal Ministry of Education and Research supports a network (LEUKONET), which aims to recruit most of the German leukodystrophy patients and, in addition, offers information for physicians, patients and relatives. All German clinical centers experienced in treating leukodystrophy patients participate in this network. The network also includes a number of basic researchers whose projects intend to elucidate the primary cause and pathogenesis of these disorders.

A new type of leukoencephalopathy with metaphyseal chondrodysplasia maps to Xq25-q27.

BACKGROUND: The authors report a three-generation family with four male patients presenting with a novel type of X-chromosomal leukoencephalopathy associated with skeletal abnormalities. METHODS: The index patient and his brother reached their early motor milestones in due time and had normal language development. Between the ages of 2 and 3 years, first signs of spastic paraplegia were noticed. Furthermore, the patients developed tremor, ataxia, optic atrophy, and spastic tetraparesis. Both boys had broad wrists and knees without significant contractures. A maternal uncle and a granduncle had the same disease. RESULTS: Leukoencephalopathy (MRI, MRS) and metaphyseal chondrodysplasia (X-ray, MRI) were diagnosed. MRS showed a reduction of choline-containing compounds in the white matter. An autopsy on one of the patients, who died at age 37 years, revealed an orthochromatic type of leukoencephalopathy. In bone and cartilage tissue, unspecific signs of a mild chondrodysplasia were found. At the PLP gene locus an obligate recombination was observed, which excludes the Pelizaeus-Merzbacher locus on Xq21-22. However, affected males share a fragment of the long arm of chromosome X. CONCLUSION: The authors report a new type of leukoencephalopathy associated with metaphyseal chondrodysplasia located on Xq25-q27.

[The role of the ophthalmologist in the management of juvenile neuronal ceroid lipofuscinosis]. / Relevanz ophthalmologischer Diagnostik für die Betreuung von Patienten mit juveniler neuronaler Ceroid-Lipofuszinose.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are storage diseases leading to severe somatic and mental deterioration with blindness and death. To date, there are no therapeutic options. Juvenile NCL (JNCL), also known as Batten's disease, is one of the most prevalent forms of NCL. MATERIALS AND METHODS: A 6-year-old boy with the primary diagnosis of retinitis pigmentosa was examined. The parents reported a rapid deterioration of vision during the past months. In view of this history, additional, non-ophthalmological diagnostic procedures have been performed (peripheral blood smear, molecular genetics). RESULTS: The eye examination showed a considerable reduction of visual acuity, a concentric visual field constriction, an extinguished electroretinogram and a bull's eye maculopathy. The peripheral blood smear revealed vacuolated lymphocytes. Molecular genetic investigation confirmed the diagnosis of juvenile NCL by detecting a homozygous (1-kb deletion of the CLN3-gene). CONCLUSIONS: The ophthalmologist plays a key role for an early diagnosis of juvenile NCL. An early diagnosis is important for the affected families because only then they can handle this stroke of fate.

Asparagine concentration in plasma after 2,500 IU/m(2) PEG-asparaginase i.v. in children with acute lymphoblastic leukemia.

UNLABELLED: Polyethylene glycol conjugated asparaginase (PEG-ASNase) can be substituted in cases of hypersensitivity to native Escherichia coli asparaginase. We measured asparagine (asn) levels in plasma after a single dose of 2,500 IU/m(2) i.v. PEG-ASNase (Oncaspar) in consolidation treatment of ALL and compared those with data from the previous protocol COALL-05-92. This protocol was similar to COALL-06-97, except that children had been given 45,000 IU/m(2) C-ASNase instead of PEG-ASNase. PATIENTS AND METHODS: Between May 2000 and December 2001 seventy-one children (38 boys, 33 girls) with newly diagnosed ALL treated according to the multicenter protocol COALL-06-97 were investigated in this study. Four hundred and seventy-four plasma samples (71 patients) were analysed by ion exchange chromatography after column derivatization with o-phthaldialdehyde. For comparison data (350 plasma samples) from 51 patients treated according to the protocol COALL-05-92 were available. The same method for detection of asn in plasma was used. RESULTS: The median asparagine level in plasma after 2,500 IU/m(2) PEG-ASNase i.v. was below the limit of detection for at least 5 weeks in 81 % of the patients. When divided into high risk (HR) and low risk (LR) group, HR patients who had previously received one dose more of C-ASNase showed a markedly shorter depletion than the LR patients compatible with a higher risk of antibody formation and consequent silent inactivation after a higher number of exposures to ASNase. In the previous protocol COALL-05-92 median asn levels in plasma after 45,000 IU/m(2) native C-ASNase i.v. were below the limit of detection for at least 5 weeks in 65 % of the patients. CONCLUSIONS: 2,500 IU/m(2) PEG-ASNase led to an equally long depletion of asn in plasma as did 45,000 IU/m(2) native C-ASNase i.v. used in COALL-05-92.

The ratio of alpha-galactosidase to beta-glucuronidase activities in dried blood for the identification of female Fabry disease patients.

Female heterozygous patients with Fabry disease are difficult to identify because of the relatively high residual activity of alpha-galactosidase. We systematically evaluated the activities of various lysosomal enzymes in dried blood samples from Fabry patients and found that the beta-glucuronidase activity was frequently elevated. The ratio of alpha-galactosidase to beta-glucuronidase proved to be a helpful tool for the diagnosis of female Fabry disease patients.

Two novel CLN6 mutations in variant late-infantile neuronal ceroid lipofuscinosis patients of Turkish origin.

Neuronal ceroid lipofuscinoses (NCLs) are the most common neurodegenerative childhood-onset disorders characterized by autosomal recessive inheritance, epileptic seizures, progressive psychomotor deterioration, visual failure, and premature death. At least seven subtypes of childhood-onset NCLs have been identified of which the late-infantile-onset forms (LINCLs) are genetically the most heterogeneous with four underlying genes identified. A variant form of LINCL (vLINCL) present in Turkish patients has been considered a distinct clinical and genetic entity (CLN7). However, we recently showed that mutations in the CLN8 gene account for a subset of Turkish vLINCL. Toward identifying the CLN7 gene we here screened the known NCL loci for homozygosity in nine Turkish vLINCL families. These loci were excluded in seven families that are likely to represent the 'true' Turkish vLINCL. In two families, we identified two novel homozygous mutations in the CLN6 gene: an intronic base substitution (c.542+5G>T) affecting the splicing of the transcript and a nonsense mutation (c.663C>G) creating a stop codon at tyrosine 221. These data indicate that CLN6 mutations, in addition to those of CLN8, should be considered a diagnostic alternative in Turkish vLINCL patients. The genetic background of the 'true' Turkish vLINCL, CLN7, remains to be defined.

Cystic leukoencephalopathy without megalencephaly: a distinct disease entity in 15 children.

OBJECTIVE: To describe a distinctive syndrome of nonprogressive encephalopathy, normo- or microcephaly, and early onset of severe psychomotor impairment in 15 white patients, including two siblings and two first cousins. METHODS AND RESULTS: MRI revealed bilateral cysts in the anterior part of the temporal lobe and white matter abnormalities with pericystic abnormal myelination and symmetric lesions in frontal and occipital periventricular regions. None of the usual inborn errors of metabolism/infectious diseases associated with leukoencephalopathy and bilateral anterior temporal lobe cysts were detected. CONCLUSIONS: These patients' clinical signs and cranial MRI abnormalities are strikingly similar and may represent a distinctive disease with autosomal-recessive inheritance: cystic leukoencephalopathy without megalencephaly.

Cumulative incidence rates of the mucopolysaccharidoses in Germany.

In 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 (beta-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.

Efficiency of long-term tetrahydrobiopterin monotherapy in phenylketonuria.

Phenylketonuria, an inborn error of phenylalanine metabolism, occurs with a frequency of about 1 in 10,000 births and is treated with a strict dietary regimen. Recently, some patients with PKU have been found to show increased tolerance towards phenylalanine intake while receiving tetrahydrobiopterin (BH(4)) supplementation. We have treated two infants with BH(4)-responsive PKU with BH(4) for more than 2 years. No additional dietary control was required to maintain blood phenylalanine concentrations in the desired range. Both children have shown normal development. Generally, our results suggest that BH(4) treatment might be an option for some patients with mild PKU, as it frees them from dietary restrictions and thus improves their quality of life.

Infantile cobalamin deficiency with cerebral lactate accumulation and sustained choline depletion.

A remarkable, intermittent sudden-onset vigilance and movement disorder in an exclusively breast-fed infant is reported, which was caused by cobalamin depletion due to maternal vitamin B12 malabsorption. The lack of cobalamin caused a severe encephalopathy in the infant, whose brain displayed a striking loss of volume and a delay of myelination. Proton magnetic resonance spectroscopy revealed an accumulation of lactate in the gray and white matter of the brain and a sustained depletion of choline-containing compounds in the white matter, reflecting a reversible disturbance of oxidative energy metabolism in brain cells and a long-lasting hypomyelination disorder. The clinical picture in conjunction with MRI and spectroscopic data of this case study yields more insight into the functions of cobalamin in the cerebral metabolism.

Towards quality assurance in the determination of lysosomal enzymes: a two-centre study.

The definitive diagnosis of lysosomal storage disorders depends on the determination of enzymatic activities in cells, tissues or body fluids. At present, neither an evaluation of the different methods nor an interlaboratory quality assurance scheme is available. We have therefore determined the activities of total hexosaminidase, hexosaminidase A and beta-galactosidase in the same samples (n = 15) at two metabolic centres in Germany. Three different enzymatic methods were employed, two of which were based on leukocytes as enzyme source and one on dried blood spots. The results obtained by the two different methods using leukocytes proved comparable. In contrast, assays with dried blood spots showed poor correlation with results from leukocytes, possibly because enzymatic activity in dried blood is mainly derived from soluble plasma proteins. Nevertheless, accurate detection of a true enzyme deficiency was also possible in dried blood spots. All enzymes were highly stable when mailed frozen (recovery 98-120%). Enzymatic activities in dried blood samples were also stable at room temperature and were not affected even by exposure to elevated temperatures (50 degrees C for 3 h). Dried blood seems to be especially well suited for mailing from distant healthcare facilities, although more accurate results can be expected from leukocytes. In summary, comparability and pitfalls within a lysosomal quality assurance programme were evaluated.

A hypothesis on the biochemical mechanism of BH(4)-responsiveness in phenylalanine hydroxylase deficiency.

We describe six children with tetrahydrobiopterin (BH(4)) responsive phenylalanine hydroxylase (PAH) deficiency. All patients carry two mutant alleles in the PAH gene. Cofactor deficiency was excluded. The effect of BH(4) administration was studied by correlating different oral BH(4) doses with plasma phenylalanine levels under defined protein intake. Our results indicate that oral BH(4) supplementation may be used as long-term treatment for individuals with BH(4)-responsive PAH deficiency, either without or in combination with a less restrictive diet. Previous in vitro studies have demonstrated that BH(4) inhibits PAH tetramers but activates PAH dimers. This may indicate, that BH(4)-responsiveness results from BH(4) induced stabilization of mutant PAH dimers. In addition, interindividual differences in the cellular folding apparatus may determine the tertiary structure and the amount of mutant PAH dimers and hence may account for divergent BH(4)-responsiveness reported for the same PAH genotype.

Altered levels of high-energy phosphate compounds in fibroblasts from different forms of neuronal ceroid lipofuscinoses: further evidence for mitochondrial involvement.

The pathogenesis of neurodegeneration in neuronal ceroid lipofuscinosis (NCL) is still not clear despite progress in mutation analysis of these diseases. We have recently observed anomalies at the level of the mitochondrial ATPsynthase (complex V of the respiratory chain) in fibroblasts from children with CLN1, CLN2, CLN3 and in an ovine model (OCL6). The measurements were carried out in vitro. If these alterations were of relevance in vivo as well, contents of high-energy phosphate compounds should be reduced. In the present study, we measured levels of creatine phosphate (CP), ATP, ADP and AMP in fibroblasts from children with CLN1, CLN2, CLN3 and in OCL6. ATP was reduced to about 50% of normal in CLN1, CLN2 and CLN3, ADP was about 30% of normal in these cells, and CP was 50% of normal in CLN1 and CLN2 but remained normal in CLN3. In fibroblasts of NCL-sheep, however, CP and ADP were increased to 690% and 220% of normal, respectively, while ATP remained normal. If the anomalies found in cellular energy metabolism in fibroblasts were expressed in neurons from NCL patients and NCL sheep 'slow-onset excitotoxicity' could occur leading to cellular dysfunction and eventually to cell death.

Cause of progression in Duchenne muscular dystrophy: impaired differentiation more probable than replicative aging.

Replicative aging of myogenic cells (satellite cells) owing to enhanced myofiber turnover is a common explanation of the progression of Duchenne muscular dystrophy (DMD). This hypothesis has been reexamined recently by telomere length measurements in dystrophic tissue. We evaluate the controversial results of these studies. We also review a large body of in vitro, animal (mdx), and patient data which indicate that impaired differentiation, but not replicative aging, is the leading cause of progression in DMD. We recommend in vivo investigations of cell kinetics in DMD muscle, as well as telomere length and telomerase analyses of DMD satellite cells in vitro for a definite judgement of the replicative aging hypothesis. Analogous investigations were helpful in AIDS research where replicative aging was embraced as a simple explanation of the paradigmatic CD4 lymphocyte decline but had to be rejected in favour of more complex models of disturbed lymphocyte homeostasis and regeneration. The question of replicative aging versus impaired differentiation is relevant for the understanding of therapeutic failures and the design of new strategies. Impaired differentiation is compatible with the failure of myoblast transfer in DMD and calls for further studies on the myofiber environment. Replicative aging, on the other hand, could possibly be treated by telomerase gene delivery.

Beneficial effects of alpha-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.