👤 Martijn H Breuning

The neuronal ceroid lipofuscinoses (NCL) are worldwide the most common lysosomal storage disorders of childhood. Clinical features often include progressive visual impairment, seizures, psychomotor deterioration, dementia, and premature death. Most NCL cases are caused by mutations in the CLN1, CLN2 and CLN3 genes, which play an essential role in lysosomal protein degradation. Laboratory diagnostics for a patient suspected of NCL should start with enzyme analysis in the case of INCL and LINCL and investigation of lymphocyte vacuolisation for JNCL. Diagnosis at the protein level is not available for JNCL, but CLN3 mutation analysis is possible. The carrier status of healthy relatives in families with known mutations in either CLN1, CLN2, CLN3 or CLN6 can be determined with certainty by mutation analysis. Show less

Multiple osteochondromas (MO) is an autosomal dominant condition, caused by mutations in either the EXT1 or the EXT2 gene. The DNA of a cohort of 35 patients, clinically suspected to be affected with MO, was screened for mutations by a combination of direct sequence analysis and multiplex ligation-dependent probe amplification (MLPA). In this cohort, 26 pathogenic gene alterations were found (74%). With sequence analysis mutations were detected in 22 patients (63%). In total, 10 mutations were detected in the EXT1 and 12 in the EXT2 gene. The number of the splice site mutations detected was larger than expected from the literature. In addition, with the MLPA four deletions of one or more exons were found in this cohort. Two patients, of whom one had a negative family history, showed deletions of exon 1 of the EXT1 gene, which is possibly a deletion hot spot. In patients suspected to be affected by MO, we recommend a quantitative analysis such as MLPA, followed by direct sequence analysis for the screening of the EXT1 and EXT2 genes. Show less

Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to detect these rearrangements, both in research and diagnostic settings. Multiplex ligation-dependent probe amplification (MLPA) is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one-tube assay. Current MLPA probe design, however, involves time-consuming and costly steps for probe generation. To bypass these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This problem was tackled by designing a two-color assay, combining two sets of probes, each amplified by primers labeled with a different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18 patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more exons: four in EXT1 and one in EXT2. Our results show that a two-color MLPA assay using only synthetic oligonucleotides provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients to be tested is limited, making it financially unattractive to invest in cloning. Show less

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL). Show less

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease. Show less

JNCL is a neurodegenerative disease of childhood caused by mutations in the CLN3 gene. A mouse model for JNCL was created by disrupting exons 1-6 of Cln3, resulting in a null allele. Cln3 null mice appear clinically normal at 5 months of age; however, like JNCL patients, they exhibit intracellular accumulation of autofluorescent material. A second approach will generate mice in which exons 7 and 8 of Cln3 are deleted, mimicking the common mutation in JNCL patients. Show less

The recent isolation of the CLN3 gene involved in Batten disease (juvenile neuronal ceroid lipofuscinosis) creates possibilities for direct detection of mutations which can confirm or indicate the clinical diagnosis of Batten disease. We have designed a rapid and reliable allele specific PCR test for the detection of the major deletion, which can be used in carrier diagnosis, presymptomatic diagnosis, and prenatal diagnosis. Show less

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. Show less

We recently cloned a cDNA for CLN3, the gene for juvenile-onset neuronal ceroid lipofuscinosis or Batten disease. To resolve the genomic organization we used a cosmid clone containing CLN3 to sequence the entire gene in addition to 1.1 kb 5' of the start of the published CLN3 cDNA and 0.3 kb 3' to the polyadenylation site. CLN3 is organized into at least 15 exons spanning 15 kb and ranging from 47 to 356 bp. The 14 introns vary from 80 to 4227 bp, and all exon/intron junction sequences conform to the GT/AG rule. Numerous repetitive Alu elements are present within the introns and 5'- and 3'-untranslated regions. The 5' region of the CLN3 gene contains several potential transcription regulatory elements but no consensus TATA-1 box was identified. CLN3 is homologous to 27 deposited human ESTs, and sequence comparisons suggest alternative splicing of the gene and the existence of transcribed sequences upstream to the start of the published CLN3 cDNA. Show less

A murine cDNA clone was isolated by screening a mouse cDNA library with the human CLN3 cDNA. Sequence analysis indicates that the corresponding CLN3 proteins are highly homologous. We have compared these with recently identified CLN3 sequences from the dog, the nematode C. elegans, and baker's yeast S. cerevisiae. The CLN3 protein is remarkably conserved across eukaryotic species. Several protein modification sites which may be crucial for the function of the protein are conserved. Show less

Accurate diagnosis of neuronal ceroid lipofuscinosis (NCL) is important for a correct prognosis of the disease and for genetic counseling. Up to now, no direct diagnostic test has been available for NCL. The clinical diagnosis is made on the basis of symptoms, neurophysiological, neuroradiological, and specific lipopigment pattern data. Recent advances in the genetics of NCL have enabled us to use polymorphic DNA markers linked to the CLN1 and CLN3 loci as a tool in the differential diagnosis of NCL. We have applied genetic analysis with polymorphic DNA markers flanking the CLN3 gene on chromosome 16 to two consanguineous families in which NCL occurs. In the first family, which is of Turkish extraction, two patients suffering from a protracted form of juvenile NCL previously had been diagnosed with juvenile NCL. Haplotypes from this family indicate that the patients and their healthy sibling are haplo-identical, suggesting that this protracted form of juvenile NCL is not linked to the CLN3 locus. In the second family, which is of Moroccan origin, one patient suffers from the early juvenile variant of NCL (Lake-Cavanagh). In this family, the patient and one of the healthy siblings have identical haplotypes, excluding linkage of early juvenile NCL to the CLN3 locus on 16p12.1-11.2. Therefore, these cases from different populations demonstrate that haplotype analysis can be used as an additional method to exclude the diagnosis of juvenile NCL. Show less

Batten disease, or the juvenile form of neuronal ceroid lipofuscinosis, is an autosomal recessive neurodegenerative disorder manifesting with progressive blindness, seizures, and dementia, leading to an early death. The CLN3 locus which is involved in Batten disease had been localized to chromosome 16p11.2. Linkage disequilibrium has been observed between CLN3 and polymorphic microsatellite markers D16S288, D16S299, and D16S298, making carrier detection and prenatal diagnosis by haplotype analysis possible. For the purpose of carrier detection, haplotypes from Dutch Batten patients and their families were constructed. Most patients share the same D16S298 allele, suggesting the presence of a founder effect in the Dutch population. In a large inbred Dutch family, in which Batten disease occurs with high frequency, haplotype analysis has been carried out with high accuracy for carrier detection. Show less

The gene that is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease--CLN3--has been localized to 16p12, and the mutation shows a strong association with alleles of microsatellite markers D16S298, D16S299, and D16S288. Recently, haplotype analysis of a Batten patient from a consanguineous relationship indicated homozygosity for a D16S298 null allele. PCR analysis with different primers on DNA from the patient and his family suggests the presence of a cytogenetically undetectable deletion, which was confirmed by Southern blot analysis. The microdeletion is embedded in a region containing chromosome 16-specific repeated sequences. However, putative candidates for CLN3, members of the highly homologous sulfotransferase gene family, which are also present in this region in several copies, were not deleted in the patient. If the microdeletion in this patient is responsible for Batten disease, then we conclude that the sulfotransferase genes are probably not involved in JNCL. By use of markers and probes flanking D16S298, the maximum size of the microdeletion was determined to be approximately 29 kb. The microdeletion may affect the CLN3 gene, which is expected to be in close proximity to D16S298. Show less

The gene for Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, or Spielmeyer-Sjögren disease), CLN3, maps to 16p11.2-12.1. Four microsatellite markers--D16S288, D16S299, D16S298, and SPN--are in strong linkage disequilibrium with CLN3 in 142 families from 16 different countries. These markers span a candidate region of approximately 2.1 cM. CLN3 is most prevalent in northern European populations and is especially enriched in the isolated Finnish population, with an incidence of 1:21,000. Linkage disequilibrium mapping was applied to further refine the localization of CLN3 in 27 Finnish families by using linkage disequilibrium data and information about the population history of Finland to estimate the distance of the closest markers from CLN3. CLN3 is predicted to lie 8.8 kb (range 6.3-13.8 kb) from D16S298 and 165.4 kb (132.4-218.1 kb) from D16S299. Enrichment of allele "6" at D16S298 (on 96% of Finnish and 92% of European CLN3 chromosomes) provides strong evidence that the same major mutation is responsible for Batten disease in Finland as in most other European countries and that it is therefore not a Finnish mutation. Genealogical studies show that Batten disease is widespread throughout the densely populated regions of Finland. The ancestors of two Finnish patients carrying rare alleles "3" and "5" at D16S298 in heterozygous form originate from the southwestern coast of Finland, and these probably represent other foreign mutations. Analysis of the number and distribution of CLN3 haplotypes from 12 European countries provides evidence that more than one mutation has arisen in Europe. Show less