CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) represent a group of common recessive inherited neurodegenerative disorders of childhood, with an incidence of 1:12,500 live births. They are characterized by accumulation of autofluorescent lipopigments in various tissues. Several forms of NCLs have been identified, based on age at onset, progression of disease, neurophysiological and histopathological findings and separate genetic loci. All types of NCL cause progressive visual and mental decline, motor disturbance, epilepsy and behavioral changes, and lead to premature death. One of the subtypes, Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCL; MIM256731) affects children at 4-7 years of age. The first symptom is motor clumsiness, followed by progressive visual failure, mental and motor deterioration and later by myoclonia and seizures. We have previously reported linkage for vLINCL on chromosome 13 (ref. 5) and constructed a long-range physical map over the region. Here, we report the positional cloning of a novel gene, CLN5, underlying this severe neurological disorder. The gene encodes a putative transmembrane protein which shows no homology to previously reported proteins. Sequence analysis of DNA samples from patients with three different haplotypes revealed three mutations; one deletion, one nonsense and one missense mutation, suggesting that mutations in this gene are responsible for vLINCL.

Head and neck arteriovenous malformations: results of ethanol sclerotherapy.

BACKGROUND AND PURPOSE: Peripheral AVM is a locally aggressive disease with a high tendency to recur; its treatment is complex, especially in the anatomically delicate head and neck area. Here, we report results of ethanol sclerotherapy for head and neck AVM and discuss its potential use for peripheral AVM. MATERIALS AND METHODS: We retrospectively assessed degree of AVM eradication, complications, and clinical or imaging signs of recurrence for 19 patients treated with ethanol sclerotherapy for head and neck AVM (1 intraosseous, 18 soft-tissue AVMs). RESULTS: Of the 19 patients, 11 had complete eradication of arteriovenous shunting at DSA, with 1 recurrence (mean follow-up 15 months), and for 7 patients, treatment is ongoing. During 59 treatment sessions, 12 patients experienced 14 complications, 1 leading to permanent functional damage. CONCLUSIONS: Ethanol sclerotherapy has potential for complete eradication of head and neck AVM with low recurrence within the first year after completion of treatment. Complete eradication may require several treatment sessions during which complications should be minimized with careful techniques.

Case report of Zenker's diverticulum in identical twins: further evidence for genetic predisposition.

OBJECTIVE: We report identical twins with Zenker's diverticulum. METHODS: Case report and literature review. CONCLUSIONS: Geographical and racial variation in occurrence, and rare familial cases, suggest that inherited factors play a role in the pathogenesis of Zenker's diverticulum. The identical twins reported here provide further evidence supporting a genetic predisposition.

Positional cloning of the CLN5 gene defective in the Finnish variant of the LINCL.

Neuronal ceroid lipofuscinoses (NCLs) in children are progressive encephalopathies inherited as autosomal recessive traits. Progressive neuronal damage leads to psychomotor deterioration, visual failure, seizures, and finally to premature death. Based on the clinical course of the disease, the childhood forms can be divided into several subtypes. A variant form of the late infantile NCL (vLINCL), characterized by mental retardation, visual failure, ataxia, myoclonia, and death between the ages of 13 and 30 years, is prevalent in Finland. Information on ancient recombination events in disease alleles rising from this isolated population provided an efficient tool for refining the initial assignment of the CLN5 locus. Here we describe the steps resulting in the identification of the novel gene, defective in vLINCL.

Utilization of FISH in positional cloning: an example on 13q22.

In positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region. The construction of a high-resolution physical map still involves considerable effort. However, new high-resolution fluorescence in situ hybridization (FISH) techniques have facilitated this process substantially. Here we summarize a strategy that combines a spectrum of FISH techniques [metaphase, interphase, mechanically stretched chromosomes (MSCs), and fiber-FISH on free chromatin] for the construction and characterization of a high-resolution physical map for a positional cloning project. The chromosomal region 13q22, containing the locus of the variant form of the neuronal ceroid lipofuscinosis (vLINCL, CLN5) disease, serves here as an example for this process. We used metaphase FISH to exclude positionally a candidate gene, to refine the locus to 13q22, and to analyze the possible chimerism of the YACs in the region. Both metaphase and interphase FISH techniques were applied to determine the low-resolution distances between the restricting markers. FISH using MSCs confirmed the centromeric-telomeric order of the clones and facilitated the estimation of the size of the gaps between the clones. Finally, fiber-FISH was found to be the method of choice for the construction of an accurate high-resolution map of the contig established over the restricted region. Thus, FISH techniques in combination with genetic mapping data enabled the refinement of the initial 4-cM region to a high-resolution map of only 400 kb in length. Here the FISH strategy replaced the need for many laborious traditional physical mapping methods, e.g., pulsed-field gel electrophoresis.

The visual assignment of genes by fiber-fish: BTF3 protein homologue gene (BTF3) and a novel pseudogene of human RNA helicase A (DDX9P) on 13q22.

Chromosome 13 is one of the poorly mapped human chromosomes. As an example, only two cloned genes have been assigned to bands 13q22-q31. Our characterization of the critical region for the variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) disease region on 13q22 resulted in the identification of the sequences encoding the BTF3 protein homologue gene (HGMW-approved symbol BTF3) and a novel pseudogene for RNA Helicase A (HGMW-approved symbol DDX9P). Precise visual assignment to the physical clones covering this region and the positional relationships of these genes were achieved by the use of tyramine enhancement of Fiber-FISH hybridization signals, demonstrating the power of this technique in efficient positioning of coding regions on the physical maps.

Transcript identification on the CLN5 region on chromosome 13q22.

Our efforts to clone the CLN5 gene, mutated in a severe children's brain disease, variant late infantile neuronal ceroid lipofuscinosis (vLINCL, MIM256731), resulted in large-scale sequencing of genomic clones flanking the critical chromosomal region on 13q22. Computational and traditional transcript identification analyses of the resulting sequence were used to identify the disease gene. In addition to the identification of the CLN5 gene, this effort produced a large amount of genomic sequence data. Here, we report a transcription map of the 107 kb sequence in the CLN5 region, based on traditional and computational gene identification strategies. Several transcripts were identified in this sequence. Queries against the database of expressed sequence tags proved to be the most powerful tool for gene identification from large-scale sequence.

Efficient construction of a physical map by fiber-FISH of the CLN5 region: refined assignment and long-range contig covering the critical region on 13q22.

The variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) represents a progressive brain disease with autosomal recessive inheritance. We have previously assigned the CLN5 locus to chromosome 13q21.1-q32 between markers D13S160 and D13S162 by linkage analysis in Finnish families. The information on ancient recombination events obtained from linkage disequilibrium provided an efficient tool for further refining the assignment of the CLN5 locus. Isolation of two novel (CA)n markers, COLAC1 and AC224, resulted in a dramatic restriction of the critical DNA region. We utilized the Fiber-FISH technique to orient and order the large DNA clones isolated by STSs and were able to eliminate almost totally the restriction digestion and PFGE step in the construction of the long-range DNA contig. Both linkage disequilibrium data and Fiber-FISH analyses assigned the CLN5 locus to a well-defined 200-kb region. Here we report a complete physical map of about 350 kb covering the critical chromosomal region of CLN5, which will facilitate the final isolation of the CLN5 gene.