Rose-Mary Boustany

Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse.

The CLN6 gene that causes variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), a recessively inherited neurodegenerative disease that features blindness, seizures, and cognitive decline, maps to 15q21-23. We have used multiallele markers spanning this approximately 4-Mb candidate interval to reveal a core haplotype, shared in Costa Rican families with vLINCL but not in a Venezuelan kindred, that highlighted a region likely to contain the CLN6 defect. Systematic comparison of genes from the minimal region uncovered a novel candidate, FLJ20561, that exhibited DNA sequence changes specific to the different disease chromosomes: a G-->T transversion in exon 3, introducing a stop codon on the Costa Rican haplotype, and a codon deletion in exon 5, eliminating a conserved tyrosine residue on the Venezuelan chromosome. Furthermore, sequencing of the murine homologue in the nclf mouse, which manifests recessive NCL-like disease, disclosed a third lesion-an extra base pair in exon 4, producing a frameshift truncation on the nclf chromosome. Thus, the novel approximately 36-kD CLN6-gene product augments an intriguing set of unrelated membrane-spanning proteins, whose deficiency causes NCL in mouse and man.

Spectrum of Mutations in the Batten Disease Gene, CLN3

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis [JNCL]) is an autosomal recessive condition characterized by accumulation of lipopigments (lipofuscin and ceroid) in neurons and other cell types. The Batten disease gene, CLN3, was recently isolated, and four disease-causing mutations were identified, including a 1.02-kb deletion that is present in the majority of patients (The International Batten Disease Consortium 1995). One hundred eighty-eight unrelated patients with JNCL were screened in this study to determine how many disease chromosomes carried the 1.02-kb deletion and how many carried other mutations in CLN3. One hundred thirty-nine patients (74%) were found to have the 1.02-kb deletion on both chromosomes, whereas 49 patients (41 heterozygous for the 1.02-kb deletion) had mutations other than the 1.02-kb deletion. SSCP analysis and direct sequencing were used to screen for new mutations in these individuals. Nineteen novel mutations were found: six missense mutations, five nonsense mutations, three small deletions, three small insertions, one intronic mutation, and one splice-site mutation. This report brings the total number of disease-associated mutations in CLN3 to 23. All patients homozygous for mutations predicted to give rise to truncated proteins were found to have classical JNCL. However, a proportion of the patients (n = 4) who were compound heterozygotes for a missense mutation and the 1.02-kb deletion were found to display an atypical phenotype that was dominated by visual failure rather than by severe neurodegeneration. All missense mutations were found to affect residues conserved between the human protein and homologues in diverse species.

Mutational Analysis of the Defective Protease in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis, a Neurodegenerative Lysosomal Storage Disorder

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3 splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Identification and Expression Analysis of Spastin Gene Mutations in Hereditary Spastic Paraplegia

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be leaky, or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

Apoptosis as the Mechanism of Neurodegeneration in Batten's Disease

Abstract: Battens disease is a genetic neurodegenerative disease of childhood. Its hallmarks are retinitis pigmentosa and neuronal degeneration. As some types of photoreceptor death in mice are mediated by apoptosis, we investigated whether apoptosis is responsible for retinal and neuronal degeneration in the late infantile and juvenile forms of Battens disease. Using the terminal dUDP nick end‐labeling (TUNEL) staining method, we detected apoptotic neuronal cells in brain from patients and a canine model and in brain and retina from an ovine model for Battens disease. We confirmed apoptosis by flow cytometry, electron microscopy, and DNA laddering. This is the first inherited neurodegenerative disease involving brain and retina in which apoptosis has been established as the mechanism of neuronal and photoreceptor cell death in both humans and animal models.

Allopregnanolone attenuates N-methyl-d-aspartate-induced excitotoxicity and apoptosis in the human NT2 cell line in culture

Progesterone modulates gamma-aminobutyric acid and excitatory amino acid neurotransmitter systems and has neuroprotective properties in models of hypoxia-ischemia. This study examined the in vitro effects of allopregnanolone, the active progesterone metabolite, in models of N-methyl-D-aspartate (NMDA)-induced necrosis and apoptosis. Cultured NT2 neurons were exposed to 1 mM NMDA. Lactate dehydrogenase (LDH) release was measured 24 h later. NMDA at a concentration of 1 mM produced a 39 +/- 19% release of total LDH. Exposure to 10 microM allopregnanolone prior to NMDA exposure reduced LDH release by 51% (P = 0.0028). NMDA stimulated apoptotic cell changes defined by terminal dUTP nick-end labeling (TUNEL) and 5,5, 6,6-tetrachloro-1,1,3,3-tetra ethlybenzimidazolycarbocyanide iodide staining were reduced to baseline values by both 10 microM allopregnanolone and 100 microM MK-801. Pretreatment with allopregnanolone (0-10 microM) reduced the percentage of TUNEL-positive cells in a dose-dependent manner (EC(50) = 2.7 +/- 0.1 nM). Physiologic concentrations of allopregnanolone provided protection against both necrotic and apoptotic injury induced by NMDA excitotoxicity.

Flupirtine blocks apoptosis in Batten patient lymphoblasts and in human postmitotic CLN3- and CLN2-deficient neurons

Multiple gene defects cause Batten disease. Accelerated apoptosis accounts for neurodegeneration in the late infantile and juvenile forms that are due to defects in the CLN3 and CLN2 genes. Extensive neuronal death is seen in CLN2‐ and CLN3‐deficient human brain as well as in CLN6‐deficient sheep brain and retina. When neurons in late infantile and juvenile brain survive, they manage to do so by upregulating the neuroprotective molecule Bcl‐2. The CLN3 gene has antiapoptotic properties at the molecular level. We show that the CLN2 gene is neuroprotective: it enhances growth of NT2 cells and maintains survival of human postmitotic hNT neurons. Conversely, blocking CLN3 or CLN2 expression in hNT neurons with adenoviral antisense‐CLN3 or antisense‐CLN2‐AAV2 constructs causes apoptosis. The drug flupirtine is a triaminopyridine derivative that acts as a nonopioid analgesic. Flupirtine upregulates Bcl‐2, increases glutathione levels, activates an inwardly rectifying potassium channel, and delays loss of intermitochondrial membrane calcium retention capacity. We show that flupirtine aborts etoposide‐induced apoptosis in CLN1‐, CLN2‐, CLN3‐, and CLN6‐deficient as well as normal lymphoblasts. Flupirtine also prevents the death of CLN3‐ and CLN2‐deficient postmitotic hNT neurons at the mitochondrial level. We show that a mechanism of neuroprotection exerted by flupirtine involves complete functional antagonism of N‐methyl‐D‐aspartate or N‐methyl‐D‐aspartate–induced neuronal apoptosis. Flupirtine may be useful as a drug capable of halting the progression of neurodegenerative diseases caused by dysregulated apoptosis.

Late onset of distinct neurologic syndromes in galactosemic siblings

We discuss siblings with galactose-1-phosphate uridyl transferase deficiency who developed neurologic complications after the age of 30. One has partial complex seizure and the other has generalized seizures, progressive ataxia, and apraxia. As more galactosemic children survive into adulthood, more neurologic complications may become more prevalent.

Clinicopathological and molecular characterization of neuronal ceroid lipofuscinosis in the Portuguese population

Abstract. A series of 53 Portuguese patients (derived from 43 families) born in the period 1963–1999 have been diagnosed with neuronal ceroid lipofuscinosis (NCL) based on clinicopathological findings. Plotting the cumulative number of new cases per year against the year of birth resulted in a slightly S-shaped curve, with a nearly straight central segment over a period of 14 years (1977–1990) indicating a continuous registration of new cases born during the corresponding time period. In this period the prevalence of overall NCL in the Portuguese population was calculated to be 1.55 per 100.000 live births.Twenty-six patients from 20 unrelated families were further evaluated by combining clinicopathological with biochemical and genetic data. No intra-familial heterogeneity was observed. Four sub-types of childhood NCL were identified: infantile NCL (INCL) with granular osmiophilic inclusions (GROD) and PPT1 deficiency (1/26), classical LINCL with curvilinear (CV) inclusions and tripeptidyl peptidase (TPP1) deficiency (3/26), variant late infantile NCL (LINCL) with fingerprint/curvilinear (FP/CV) inclusions and normal TPP1 enzyme activity (11/26) and juvenile NCL (JNCL) with a mix of FP/CV (11/26). Eight of 11 JNCL patients were homozygous for the 1.02-kb deletion in the CLN3 gene, and 3 were heterozygous with an unidentified mutation in the second allele. The 1.02-kb deletion in the CLN3 gene accounted for 86.3 % (19/22) of CLN3-causing alleles and 36.5 % (19/52) of childhood NCL defects. The causal mutations for CLN1 and CLN2 were V181M (2/2) and R208X (4/6), respectively. CLN1, CLN2 and CLN3 affected 3.8 %, 11.5 % and 42.3 % of NCL Portuguese patients, respectively. In 42.3 % of patients affected by the vLINCL form, CLN3, CLN5 and CLN8 gene defects were excluded by direct sequencing of cDNA. Genetic variants such as CLN6 might therefore cause a significant portion of childhood NCL in the Portuguese population.The relative frequency of classical childhood forms of NCL in the Portuguese population is reported and contributes to the knowledge of genetic epidemiology of these world-widely distributed disorders.

Ultrastructural findings in skin from patients with Niemann-Pick disease, type C

Niemann-Pick disease, type C is a lysosomal storage disease characterized by hepatosplenomegaly, the presence of foam cells in the reticuloendothelial system, and gradual motor and cognitive decline leading to death. Over 90% of patients demonstrated a defect of cholesterol esterification in cultured fibroblasts. This finding allows a reliable biochemical diagnosis; however, the test is complex and time-consuming and only available in a few centers. Ultrastructural examination of skin biopsy in 5 patients with Niemann-Pick disease, type C demonstrated lysosomes containing loosely arrayed dark lamellated structures within a clear matrix. Affected cells included macrophages, axons, pericytes, Schwann cells, smooth muscle cells, and fibroblasts with relative sparing of vascular endothelium and melanocytes. These findings demonstrate the usefulness of this simple and readily available morphologic test for the diagnosis of Niemann-Pick disease, type C.