The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative lysosomal storage disorders caused by the accumulation of ceroid and lipofuscin in various cell types, mainly cells of the cerebral cortex, cerebellar cortex, and retina (Dyken et al. 1988; Williams and Mole 2012). Characteristic features at onset include clumsiness; deterioration of vision and psychomotor functions; seizures and behavioral changes. Progression of clinical features results ultimately in total disability, blindness and premature death. Although NCL affects primarily children, age of onset of symptoms varies from infancy to adulthood. The incidence of NCL is variable and ranges from 1.3 to 7 per 100,000 (Mole and Williams 2013). However, it is more common in the northern European populations, particularly Finland where the incidence may reach 1 in 12,500 individuals and a carrier frequency of 1 in 70 (Rider and Rider 1988; Vesa et al. 1995). NCLs are clinically and genetically heterogeneous. A nomenclature and classification based both on the age of onset of symptoms and the disease-causing gene has been recently developed, which classifies NCLs into thirteen subtypes (CLN1-8, 10-14) (Williams and Mole 2012). The causative gene for the CLN9 phenotype has not been identified yet (Schulz et al. 2004).
Of note, NCLs were previously known as Batten disease. However, in recent nomenclature, Batten disease only applies to NCL caused by mutations in CLN3. CLN8 is further divided into two subgroups based on the age of onset, disease course, and severity of symptoms.
1-CLN8 disease, late infantile variant, is characterized by onset between 2-7 years of age and rapid course. Symptoms at onset include speech delay, ataxia, seizures, myoclonus, and visual and psychomotor decline. Most patients are wheelchair-bound within a few years of disease onset (Ranta et al. 2004; Topcu et al. 2004). This form of NCL was designated as Turkish variant late infantile NCL because it was first described in Turkish patients (Mitchell et al. 2001).
2-CLN8 disease, EPMR (Progressive Epilepsy with Mental Retardation), also known as Northern epilepsy, is characterized by onset between 5-10 years of age and survival prolonged into the fifth decade of life. Epilepsy, marked by tonic-clonic seizures that are resistant to drugs, is the first symptom, followed by mental retardation. Additional features include behavioral changes, mainly during puberty; loss of control of fine motor skills, and balance difficulties. Visual impairment was reported in some cases. The disease course is usually less severe than that of the late infantile variant. The vast majoritiy of patients affected with EPMR are from Northern Finland (Hirvasniemi et al. 1994; Ranta and Lehesjoki, 2000).
Most CLNs are inherited in an autosomal recessive manner. Both forms of CLN8 are caused by pathogenic variants in the CLN8 gene and are autosomal recessive.
A CLN8- founder mutation, (c.70C>G; p.Arg24Gly), with a carrier frequency of 1:135 was documented to be the cause of EPMR in patients from Finland (Ranta et al. 1999; Ranta et al. 2000).
CLN8 disease, late infantile variant, is caused by other pathogenic variants in CLN8, which were first documented in Turkish families (Ranta et al. 2004). More recent data indicate that this from of NCL is pan-ethnic. About 25 CLN8 pathogenic variants, mainly of the missense type, have been reported. A few small deletions, two of which are in-frame; and a large deletion have also been reported. CLN8 pathogenic variants were documented in patients from diverse ethnic and geographic origins such as Italy, Israel, Germany, and Pakistan (Cannelli et al. 2006; Zelnik et al. 2007; Reinhardt et al. 2010).
The CLN8 gene encodes a transmembrane protein which is hypothesized to be involved in cell survival (Vantaggiato et al. 2009).
This test involves bidirectional DNA Sanger sequencing of all coding exons and splice sites of CLN8. The full coding sequence of each exon plus ~ 20 bp of flanking DNA on either side are sequenced. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.
Candidates for this test are patients with a clinical diagnosis of CLN8 disease, variant late infantile, regardless of their ethnic or geographical origins; and patients with EPMR.
|Offical Gene Symbol||OMIM Id|
|Ceroid Lipofuscinosis Neuronal 8, Northern Epilepsy Variant||610003|
|Ceroid Lipofuscinosis Neuronal 8||600143|
|Test Number||Test||Price||CPT Code|
|848||CLN8 Sanger Sequencing||$580||81479|
|100||CLN8 Targeted Familial Mutations - Single Exon Sequencing||$250||81479|
|200||CLN8 Targeted Familial Mutations - Double Exon Sequencing||$370||81479|
|300||CLN8 Targeted Familial Mutations - Triple Exon Sequencing||$440||81479|
As required, DNA is extracted from the patient specimen using a 5 Prime ArchivePure DNA Blood Kit. PCR is used to amplify the indicated exons plus additional flanking intronic or other non-coding sequence. After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In most cases, sequencing was performed in both forward and reverse directions; in some cases, sequencing was performed twice in either the forward or reverse directions.
This test will detect pathogenic variants in the CLN8 gene in up to 95% of patients, with clinical diagnosis of CLN8 disease, late infantile variant, of various ethnicity; and in nearly all patients with EPMR (Mole and Williams 2013).
As of November 2014, we compared 11.3 megabases of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 11 years of our lab operation we have Sanger sequenced roughly 4,000 PCR amplicons (~ 2 megabases). Only one error was identified.
Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).
In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.
Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.
In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.
In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.
Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.
Maximum of 30 days, although many tests are completed in 2-3 weeks.
Last Updated 01/22/2014