Terry J. Lerner

ISOLATION OF A NOVEL GENE UNDERLYING BATTEN-DISEASE, CLN3

Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomotor disturbances. The Batten disease gene, CLN3, maps to chromosome 16p12.1. The so-called 56 chromosome haplotype defined by alleles at the D16S299 and D16S298 loci is shared by 73% of Batten disease chromosomes. Exon amplification of a cosmid containing D16S298 has yielded a candidate gene that is disrupted by a 1 kb genomic deletion in all patients carrying the 56 chromosome. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the candidate as the CLN3 gene. The disease gene encodes a novel 438 amino acid protein of unknown function.

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.

Chromosomal localization of two genes underlying late-infantile neuronal ceroid lipofuscinosis

ABSTRACT Classical late-infantile neuronal ceroid lipofuscinosis (LINCL;CLN2) is an inherited neurodegenerative disorder of childhood characterized by seizures, loss of vision, and progressive motor and mental deterioration. The hallmark of this disease is the accumulation of enlarged, secondary lysosomes packed with curvilinear bodies in cells of affected individuals. The biochemical basis of LINCL remains unknown and there is no treatment effective in delaying the progression of this fatal disorder. During a genome-wide search using a set of highly polymorphic markers and 15 affected individuals from 7 multi-affected families, we obtained evidence for linkage of the LINCL gene CLN2 with markers on chromosome 11p15.5. We then genotyped patients and all available family members, including 8 single-affected families, for markers spanning 15 cM of 11p15.5. We obtained a maximum two-point LOD score of 6.16 at θ = 0.00 at the marker locus D11S2362. Multipoint analysis yielded a maximum LOD score of 6.90 localized to the same marker. Using haplotype analysis, we localized CLN2 to a minimum candidate region of 11 cM flanked by marker loci D11S4046 on the telomeric side and D11S1996 on the centromeric side. Additionally, we present data suggesting that the gene underlying a variant LINCL subtype found in Costa Rica maps to the region defined by the CLN6 locus on chromosome 15q21-23. The mapping of these two LINCL loci provides a genetic basis for understanding the clinical heterogeneity observed in this group of diseases.

Peripherin gene is linked to keratin 18 gene on human chromosome 12

Peripherin is a neuron-specific intermediate filament (IF) protein, found primarily in phylogenetically old regions of the nervous system. Whereas other neuronal IF genes have only two to three introns and are scattered in the genome, the peripherin gene (PRPH) has a complex intron-exon structure like nonneuronal IF genes that are clustered in tandem arrays, e.g., those encoding the keratins. We used a cosmid containing the human peripherin gene (PRPH) to determine its chromosomal location in relationship to nonneuronal IF genes. Using a rodenthuman mapping panel, we localized thePRPH gene to human chromosome 12. Since a cluster of keratin genes maps to 12q12–13, polymorphic markers were developed forPRPH and for one of the keratin genes presumed to be in the cluster, keratin 18 (KRT18). Both markers were typed in CEPH reference families. Pairwise and multipoint analyses of the CEPH data revealed thatKRT17 is tightly linked to DNA markersD12S4, D12S22, D12S90, D12S96 andD12S103, which lie betweenD12S18 andD12S8, with odds greater than 1000∶1. These markers are physically located at 12q11–13, thus supporting the fine localization ofKRT18 in or near the group of type II keratins in this region. Furthermore, linkage analysis showed that the peripherin gene (PRPH) is tightly linked toKRT18 (Ž=15.73, θ=0.013), and therefore appears to be in close proximity to the cluster.

5 Positional cloning of the JNCL gene, CLN3

Publisher Summary Juvenile neuronal ceroid lipofuscinosis (CLN3, JNCL, and Batten or Spielmeyer-Vogt-Sjogren disease, MIM304200) is the most common neurodegenerative disorder of childhood. Inheritance of the disease is autosomal recessive. There is no cure for JNCL. Treatment is largely symptomatic. Eiberg first mapped the JNCL locus CLN3 to chromosome 16. It is discovered a previously unreported subunit 9 gene, P3, and demonstrated the mapping of this gene to chromosome 2, also excluding it as the JNCL gene. Refined localization of CLN3 only became possible with the advent of highly polymorphic microsatellite markers. In parallel to efforts to refine the genetic locus of CLN3, a physical map consisting of yeast artificial chromosomes (YACs) and cosmid contigs is generated to span the candidate region. Cosmid clone NL11A, the most likely site of the JNCL gene CLN3, is selected for exon trapping for the isolation of candidate JNCL gene transcripts. The common mutation in JNCL is a 1-kb genomic deletion. The finding of additional mutations of the gene encoding cDNA2-3 in JNCL patients supported the conclusion that this gene is the JNCL gene CLN3. The tissue expression of CLN3 reflects the expression pattern of the other NCL genes so far identified, and offers no clue toward understanding the neuronal specificity of this group of disorders. No homology has been found at either the nucleotide or the protein level with any known gene, indicating that CLN3 encodes a novel protein of unknown function. The chapter discusses the animal models of JNCL. These animal models are critical for studies of the pathogenesis underlying the disease and for the evaluation of therapies.

Positional cloning of disease genes on chromosome 16

The project seeks to elucidate the molecular basis of an important genetic disease (Batten`s disease) by molecular cloning of the affected gene by utilizing an overlapping clone map of chromosome 16. Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressive loss of vision, seizures, and psychomoter disturbances. The Batten disease gene was genetically mapped to the chromosome region 16p 12.1 in close linkage with the genetic markers D16S299 and D16S298. Exon amplification of a cosmid containing D16S298 yielded a candidate gene that was disrupted by a 1 kb genomic deletion in all patients containing the most common haplotype for the disease. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the gene as the Batten disease gene. The disease gene encodes a novel 438 amino acid membrane binding protein of unknown function.