Background
The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by epilepsy, progressive cognitive and motor decline, loss of vision, dementia, and usually reduced life expectancy [
1]. NCL has been recognized as one of the most frequent childhood-onset neurodegenerative pathologies, with a prevalence of 1:1,000,000 to 1:14,000 worldwide [
2]. NCLs are classified into six subtypes according to the primary onset of symptoms, and broad phenotypic variance has been reported in different subtypes [
3]. The accumulation of autofluorescent lysosomal storage material in the central nervous system is a key pathological finding of NCLs. Several possible candidate genes are involved in this process, including genes encoding lysosomal enzymes (
CLN1/PPT1, CLN2/TPP1, CLN10/CTSD, CLN13/CTSF), genes encoding a soluble lysosomal protein (
CLN5) and genes encoding a protein in the secretory pathway (
CLN11/GRN). In addition, genes encoding cytoplasmic proteins (
CLN4/DNAJC5,
CLN14/KCTD7) and transmembrane proteins (
CLN3,
CLN6,
CLN7/MFSD8,
CLN8,
CLN12/ATP13A2) are also associated with NCLs [
4]. In addition to phenotypic and genetic heterogeneity, allelic heterogeneity of the same gene has also been previously described in affected patients [
5], indicating that precise diagnosis of NCLs may mainly rely on molecular genetic testing.
To date, more than 430 pathogenic variants in the above 13 candidate genes have been reported in human NCLs, and most have been registered in the NCL Mutation Database (
http://www.ucl.ac.uk/ncl/) [
6]. Seventy-four patients were attributed to pathogenic variants in the
CLN8 gene (OMIM #607837). Sporadic NCL patients have been occasionally reported in Chinese [
7‐
9], however, the variant spectrum in Chinese NCL patients is still unknown. In addition, variants in
CLN8 have not been identified in Chinese NCL patients. Here we reported our diagnostic experience of a Chinese boy presenting typical clinical manifestation of NCL. We identified two novel pathogenic variants in
CLN8 using targeted next-generation sequencing and completed genetic diagnosis for this patient in a very short turn-around time. To the best of our knowledge, this is the first reported NCL patient due to
CLN8 variants in China. Our report demonstrates the absolute diagnosis advantage of high-throughput genomic sequencing for pediatric neurodegenerative disease with high phenotypic and genetic heterogeneity, making patients move to the precise genotyping.
Discussion and conclusions
NCLs are a group of heterogeneous disorders characterized clinically by visual failure, behavioral problems, onset of seizure, cognitive regression and motor impairment. For the clinician, the key suggestive sign for NCLs is electron microscopy examination showing ultra-structural lipofuscinic pigments [
11]. Reduced enzyme activity is another suggestive sign for some subtypes of NCLs [
12]. Despite these clinical clues, NCLs remain a challenge for neurologists, especially for the pediatric neurologist, because the clinical signs in young children or toddlers are subtle and often overlap with other congenital neurodegenerative diseases, such as mitochondrial disorders or early-onset Parkinsonism [
11]. For the geneticist, the variants in different candidate genes could cause the same NCL subtype. Meanwhile, variants in the same candidate gene (but in different alleles) could cause different NCL subtypes [
5]. Thus, screening all relevant disease-related genes is an efficient and simple procedure to achieve precise diagnosis.
In this study, we reported a Chinese patient with NCL caused by two novel null variants in
CLN8. Both variants are interpreted as “pathogenic” according to the guidelines of ACMG. These two variants are located close to the transmembrane residues and predicted to cause invalid proteins due to the truncated transmembrane domain. To our knowledge, this is the first reported Chinese NCL patient with
CLN8 variants and the second reported Asian NCL patient with
CLN8 variants [
13]. In general, the variants in
CLN8 are associated with two different phenotypes: (1) the EPMR phenotype (MIM#610003, epilepsy with progressive mental retardation, also called as Northern Epilepsy), which is characterized by normal early development, onset of drug resistance and generalized tonic-clonic seizure between the ages of 5 and 10 years; visual loss is not a prominent feature [
14,
15]; and (2) late-infantile NCL (LI-NCL) phenotype, in which the onset of seizure is earlier and the disease progression is more rapid than EPMR [
16‐
18]. This Chinese patient had suffered from intractable epilepsy after 4 years old, and his cognitive function and vision have progressively deteriorated after the epilepsy attack. Combining the phenotype and genetic testing results, the patient in our study was precisely diagnosed as variant LI-NCL (vLI-NCL).
So far, 74 NCL patients have been attributed to the pathogenic variants in
CLN8, and most patients are from Finland and Turkey. The most frequent variants in
CLN8 were missense variants. The variants carried by the patient of our study were two novel null mutations (stop-gain), which have never been reported. The correlation between the phenotypic severity and variant category of
CLN8 remains unclear. However, the discoveries from patients with
CLN6 variants have provided some clues. In general, the null variant of
CLN6 results in severe clinical symptoms that occurs in the late infantile years, presenting with seizure, followed by severe vision loss, ataxia, mental regression and early death [
5,
19]. In contrast, the missense variant results in a milder and early-adult-onset form characterized by progressive myoclonic epilepsy alone without visual failure [
20]. We reviewed the NCL Mutation Database (
http://www.ucl.ac.uk/ncl/) and the updated literature to analyze the phenotype-variant correlation for the NCL patients with
CLN8 variants [
17,
21‐
24]. We found that the patients carrying
CLN8 null variants presented earlier onset and more progressive disease course than the patients carrying
CLN8 missense variants. We also reviewed the clinical phenotype of patients carrying
CLN8 bi-allelic or uni-allelic null variants. As Table
1 shows, seven NCL patients carrying null
CLN8 variants were retrieved and three of them carried bi-allelic null variants. All are Western patients and presented with LI-NCL or vLI-NCL, not EPMR. Our Chinese patient also suffered from intractable epilepsy, progressive vision loss, cognitive impairment and dysphagia, which is similar to that of Western patients. In addition, we found that variants in different domains of CLN8 protein cause same phenotypic severity of NCLs, suggesting that the
CLN8 variant does not have allelic heterogeneity for the NCL phenotype. We presumed that modifier variants in other NCL candidate genes or variants in the whole genome may be involved in the phenotypic variability of NCLs.
Table 1
The NCL phenotypes/subtypes in patients carrying CLN8 null variants
Pa-CLN8.033 | c.88delG (p.Ala30fsX20) | c.88delG (p.Ala30fsX20) | vLI-NCL | 3y | CL/FP | Turkey | |
Pa-CLN8.035 | c.66delG (p.Gly22SerfsX5) | c.581A > G (p.Gln194Arg) | vLI-NCL | 4y | CL/FP | Italy | Cannelli et al., 2006 [ 22] |
Pa-CLN8.036 | c.66delG (p.Gly22SerfsX5) | c.473A > G (p.Tyr158Cys) | vLI-NCL | 3.5y | CL/FP | Italy | Cannelli et al., 2006 [ 22] |
Pa-CLN8.043 | c.544-2566_590del2613 (p.Ala182AspfsX49) | c.544-2566_590del2613 (p.Ala182AspfsX49) | vLI-NCL | 2.5y | CL/FP | Turkey | Reinhardt et al., 2010 [ 17] |
Pa-CLN8.068 | c.562_563delCT (p.Leu188ValfsX58) | 8p23.3 terminal deletion | vLI-NCL | 4y | FP (lymphocytes), CL, RL (skin) | Ireland | |
Pa-CLN8.073 | c.763C > T | 8p23.3 deletion, 235 Kb | vLI-NCL | NA | NA | UK | R. Williams pers. comm |
Pa-CLN8.074 | c.728 T > C | 8p23.3 deletion, 54 Kb | vLI-NCL | NA | NA | UK | R. Williams pers. comm |
This study | c.298 C > T (p.Gln100Ter) | c.551 G > A(p.Trp184Ter) | vLI-NCL | 4y | NA | Chinese | |
In the past, candidate genes were tested one by one in the clinical diagnosis laboratory. However, this approach is time and labor consuming. With the broad development of next-generation sequencing techniques, target panel sequencing and whole exome/genome sequencing have been accepted as efficient and affordable approaches for genetic diagnosis in Mendelian recessive diseases and are particularly suitable for pediatric neurodegenerative diseases with high phenotypic and genetic heterogeneity, such as NCLs [
25]. Previous studies have also demonstrated that target panel sequencing and whole exome sequencing are appropriate methods for undiagnosed pediatric neurodevelopmental disorders [
26‐
28]. The primary results from the Epi4K/EuroEPINOMICS study revealed that trio-based next-generation sequencing provided a clear genetic etiologic diagnosis for approximately 12% of 356 patients with epilepsy [
26]. In addition, whole exome sequencing can identify causative variants in 24–33% cases with unknown intellectual disability [
27,
28]. In this study, the NCL candidate gene panel sequencing was chosen for the clinical suspicious NCL patient. The molecular diagnosis was reached within a very short turn-round time (3 weeks), suggesting that targeted sequencing is a powerful and efficient approach for clinical genetic laboratories to rapidly determine the molecular basis of NCLs and other analogous developmental disorders.
This study described a Chinese NCL patient who was diagnosed by targeted next-generation sequencing. This is the first reported Chinese patient presenting vLI-NCL phenotypes caused by two novel null variants in CLN8. Our findings expanded the variant diversity of CLN8 and proved the utility value of targeted next-generation sequencing for pediatric NCLs.