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Mutations in CLN7/MFSD8 are a common cause of variant late-infantile neuronal ceroid lipofuscinosis

Maria Kousi, Eija Siintola, Lenka Dvorakova, Hana Vlaskova, Julie Turnbull, Meral Topcu, Deniz Yuksel, Sarenur Gokben, Berge A. Minassian, Milan Elleder, Sara E. Mole, Anna-Elina Lehesjoki
DOI: http://dx.doi.org/10.1093/brain/awn366 810-819 First published online: 6 February 2009


The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881C>A (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined.

  • CLN7
  • MFSD8
  • mutations
  • neuronal ceroid lipofuscinosis


The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders encountered in childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons, but also in extraneural cells (Haltia, 2003; Mole et al., 2005). NCLs are inherited disorders that in most cases show a recessive mode of inheritance. Clinically, they present with epileptic seizures, myoclonus, progressive mental and motor deterioration, and visual failure. They usually lead to a vegetative state and eventually to premature death (Haltia, 2003).

Five forms of NCLs are recognized based on the age of onset, clinical features and the ultrastracture of the storage material. These are the congenital (CLN10 [MIM 610127]), infantile (INCL; CLN1 [MIM 256730]), late-infantile (LINCL; CLN2 [MIM 204500]), juvenile (JNCL; CLN3 [MIM 204200]) and adult (ANCL; CLN4 [MIM 204300]) NCL forms (Mole et al., 2005). In addition, several variant forms are recognized today. The variant LINCL (vLINCL) forms include at least four entities: CLN5 ([MIM 256731]; Santavuori et al., 1982), CLN6 ([MIM 601780]; Sharp et al., 1997), CLN7 ([MIM 610951]; Wheeler et al., 1999) and CLN8 ([MIM 600143]; Ranta et al., 2004; Cannelli et al., 2006). Northern epilepsy (progressive epilepsy with mental retardation, EPMR; CLN8) represents a well-described juvenile-onset phenotypic variant of CLN8 (Hirvasniemi et al., 1994) while CLN9-deficient NCL (MIM 609055) is a variant form of juvenile NCL (Schulz et al., 2004).

Eight genes underlying NCLs have been identified. These can be broadly divided into two groups: (i) the genes encoding lysosomal enzymes and (ii) the genes encoding membrane proteins of unknown function. The CLN1/PPT1 and CLN2/TPP1 genes that underlie INCL and classical LINCL encode the lysosomal enzymes palmitoyl-protein thioesterase 1 (PPT1) and tripeptidyl peptidase I (TPP1), respectively (Vesa et al., 1995; Sleat et al., 1997). Defects in a lysosomal proteinase, cathepsin D (CTSD), encoded by CTSD/CLN10, were recently shown to underlie congenital and later-onset forms of NCL (Siintola et al., 2006; Steinfeld et al., 2006; Fritchie et al., 2008). Other NCL genes (CLN3, CLN5-CLN8) code for proteins that localize either to the lysosomes or to the endoplasmic reticulum (ER). CLN3 codes for the lysosomal membrane protein CLN3 (International Batten Disease Consortium, 1995; Järvelä et al., 1998), CLN5 for the glycosylated soluble lysosomal protein CLN5 (Isosomppi et al., 2002; Holmberg et al., 2004), and the recently identified major facilitator superfamily domain containing eight (CLN7/MFSD8) gene for a putative lysosomal transporter, MFSD8 (Siintola et al., 2007). CLN6 encodes a membrane protein localizing to the ER (Gao et al., 2002; Wheeler et al., 2002; Mole et al., 2004; Heine et al., 2004), and CLN8 a membrane protein localizing both to the ER and the ER-Golgi intermediate compartment (Ranta et al., 1999; Lonka et al., 2000). CLN4 and CLN9 are provisional assignments for loci and genes that remain to be identified.

The clinical presentation of Turkish vLINCL is similar to other vLINCL forms (Topcu et al., 2004). The intracellular storage material is characterized by the presence of condensed fingerprint profiles in lymphocytes and fibroblasts (Topcu et al., 2004). Turkish vLINCL was originally thought to represent a distinct clinical and genetic entity, CLN7 (Wheeler et al., 1999), but is now known to be genetically heterogeneous with at least three underlying genes. These include CLN6 (Siintola et al., 2005), CLN8 (Ranta et al., 2004), and the recently identified CLN7/MFSD8 gene, the identification of which was based mainly on Turkish patients (Siintola et al., 2007).

Here we evaluated the molecular genetic background in 119 vLINCL patients from 112 families. We report eight novel CLN7/MFSD8 mutations and clinical findings in patients from several ethnic origins. We also report novel mutations in CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8. Known human NCL loci were excluded by haplotype and sequencing analyses in 36 Turkish patients that are likely to represent novel vLINCL forms.

Materials and Methods

Patients and controls

Overall, 71 patients from 67 Turkish families, and 48 patients from 45 families of non-Turkish origin were analysed. All had received a diagnosis of vLINCL. Clinical data were obtained from medical chart reviews. DNA was extracted by standard techniques either from leukocytes or cultivated fibroblasts. For patient 450Pa and a control individual, total RNA was extracted from fibroblasts. All samples were collected after an informed consent was obtained, according to the Declaration of Helsinki.

The control panel for CLN7/MFSD8 mutations composed of 200 Turkish or 200 Indian control chromosomes. For the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 mutations, the control panel composed of 122 Turkish and 100 CEPH (Centre d’ Etude du Polymorphisme Humain) control chromosomes. This study was approved by an Institutional Review Board of the Helsinki University Central Hospital and by an Ethics Committee of the Central Faculty Hospital in Prague and University College London.

Haplotype analysis of known human NCL loci

Homozygosity was evaluated over the CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8 and CLN10 loci using three to four fluorescently labelled microsatellite markers as previously described (Siintola et al., 2005, 2007).

Mutation screening

The exons and exon–intron boundaries of the CLN1/PPT1, CLN2/TPP1, CLN3, CLN5, CLN6, CLN7/MFSD8 and CLN8 genes were screened for mutations by genomic sequencing. For five patients screened for CLN7/MFSD8 mutations, the genotype was deduced from parents’ sequences. Polymerase chain reaction (PCR) conditions and primer sequences are available as Supplementary data (Tables S1–S7). Sequencing of the purified PCR products was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3730 DNA Analyzer or an ABI Prism 3100-Avant (Applied Biosystems). The Sequencher 4.8 program (Genes Codes Corporation, Ann Arbor, MI, USA) or Sequencing Analysis 5.1 program (Applied Biosystems) was used for sequence analysis. Patient N0803 was additionally screened for the prevalent CLN3 mutations c.461-677del and c.791-1056del (International Batten Disease Consortium, 1995) in the HUSLAB Molecular Genetics Laboratory, University of Helsinki. The control chromosomes were screened for the identified mutations by genomic sequencing.

Analysis of the c.63-4delC change

To evaluate the consequence of the c.63-4delC alteration in CLN7/MFSD8, RNA from patient 450Pa and from a control fibroblast cell line were reverse transcribed using random hexamer primers, oligo(dT), and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). Portions of the cDNA were subsequently amplified using the following forward: 5′-CCTTCAGTCCTGGCTCTGAC-3′ and 5′-GCTCTTAGGCGACACACCTG-3′, and reverse: 5′-AGCAATAACCCAGCCCAAA-3′, 5′-CACACCTTTTTCTCCAAGGAA-3′ and 5′-GGGAACCTGAGCTTCATCTG-3′ exonic primers annealing to exons 1, 2, 5, 7 and 9, respectively.


MFSD8 protein orthologues were identified using NCBI protein–protein BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and the peptide sequences were aligned using the version 5.8 of the MAFFT programme (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/). For the prediction of the possible impact of the missense mutations identified, the prediction programs PolyPhen (http://genetics.bwh.harvard.edu/pph/), SNPs3D (http://snps3d.org/), and SIFT (http://blocks.fhcrc.org/sift/SIFT.html) were used.


Haplotype analysis in Turkish patients

Given that most Turkish patients were consanguineous, we searched for homozygosity over the known human NCL loci in a subset of 61 Turkish patients. The number of patients that showed homozygous marker alleles in haplotype analysis was nine for CLN1, four for CLN2, one for CLN3, one for CLN5, 12 for CLN6, eight for CLN7 and 18 for CLN8 (data not shown). The patients were screened for mutations in the corresponding genes by sequencing (see below).

Novel mutations in CLN7/MFSD8

CLN7/MFSD8 was screened for mutations in a total of 80 patients belonging to 75 families. Nine novel sequence variants were identified. Eight of these (in 25 patients from 22 families) were likely to be disease-associated mutations (Table 1 and Fig. 1). In addition, the previously identified c.754 + 2T>A and c.929G>A (p.Gly310Asp) mutations (Siintola et al., 2007) were identified in seven patients.

Figure 1

Sequence chromatograms showing the CLN7/MFSD8 mutations identified in this study. For each mutation the control (C) sequence is shown above the mutation sequence. The mutations are highlighted by a red line underlying the affected nucleotide, with two exceptions: for c.627_643delGTATACAACACCAGTTT the 17 bases that are deleted are underlined in the control sequence, and for the heterozygous c.1103-2delA mutation, the altered intronic sequence is underlined and given in italics under the control sequence. The exon–intron boundaries are shown with black vertical lines. The arrow in the sequence of patient 397Pa shows the position at which the 17 bases have been deleted. All sequencing chromatograms are shown in the forward orientation.

View this table:
Table 1

Novel CLN7/MFSD8 mutations

Nucleotide changePredicted amino acid change/ consequenceLocationPatient codeNumber of families with the mutationCountry of origin
c.103C>Tp.Arg35XExon 3270Pa1Turkey
c.416G>Ap.Arg139HisExon 5PP1India
c.468_469delinsCCp.Thr156_Ala157delins Thr156_Pro157Exon 6475Pa1The Netherlands
c.627_643 delGTATA CAACACCAGTTTFrameshift and premature stop; p.Met209IlefsX3Exon 7397Pa1Italy (Sardinia)
c.863+1G>CSplicing defectIntron 9N34031Turkey
c.881C>Ap.Thr294LysExon 10447Pa, N3703 and N37042Turkey
367Pa, 367Pb, 380Pa, 427Pa, 466Pa, 6, 7, 8, 9, 10, 12, 14, 15 and 1812Roma from the former Czechoslovakia
161Czech Republic
c.1103-2delASplicing defectIntron 11111Czech Republic
c.1393C>Tp.Arg465TrpExon 13474Pa1Albania/Greece

Patient PP, of Indian origin, was homozygous for the missense mutation c.416G>A resulting in an arginine to histidine substitution at position 139 (p.Arg139His). The missense mutation c.881C>A was identified in homozygous form in three patients of Turkish origin, one patient of Czech origin and 14 Roma patients from the former Czechoslovakia. This nucleotide change results in a threonine to lysine substitution (p.Thr294Lys). Patient 474Pa of Greek/Albanian origin was found to be homozygous for a third missense mutation (c.1393C>T) leading to an arginine to tryptophan change (p.Arg465Trp). The p.Arg139His and p.Arg465Trp mutations affect highly conserved amino acids (data not shown) at the fourth and the eleventh putative transmembrane domains, respectively. The p.Thr294Lys mutation affects an amino acid located in the predicted seventh lumenal loop of the protein that is conserved in 11 out of 12 vertebrate and invertebrate species analysed; in zebrafish only, a serine exists instead of a threonine (data not shown). All three missense changes were indicated as probably deleterious, according to the prediction programs PolyPhen, SNPs3D and SIFT.

A nonsense mutation (c.103C>T) was identified in homozygous form in patient 270Pa, who is of Turkish origin. It creates a premature stop codon at position 35 (p.Arg35X), and predicts a truncation of the protein by 483 amino acids. Patient N3403 of Turkish origin was homozygous for a G to T transversion at the first nucleotide of intron 9 (c.863 + 1G>C), potentially affecting the splicing of the transcript. Patient 11 of Czech origin was compound heterozygous for the previously described c.754 + 2T>A mutation and a novel putative splice-site mutation, a deletion of an adenine at the penultimate nucleotide of intron 11 (c.1103-2delA). RT-PCR analyses to confirm the impact of either c.863 + 1G>C or c.1103-2delA on splicing were not possible, since patient RNA samples were not available.

Patient 475Pa of Dutch origin was homozygous for the c.468_469delinsCC deletion/insertion mutation, resulting in the in-frame substitution of an alanine at position 157 with a proline (p.Thr156_Ala157delinsThr156_Pro157). This in-frame deletion/insertion affects an alanine residue located at the fourth cytosolic loop of the protein that is highly conserved across vertebrates. Finally, in patient 397Pa of Sardinian origin, a 17bp deletion (c.627_643delGTATACAACACCAGTTT) was detected in homozygous form, resulting in a frameshift and insertion of a premature stop codon (p.Met209IlefsX3), and predicted to truncate the protein by 307 amino acids.

A ninth sequence variant, c.63-4delC, at the 3′ end of intron 2, was identified in heterozygous form in patient 450Pa of Polish origin. The same change was identified in heterozygous form in the patient's father. RT-PCR analysis from patient RNA showed no aberrations associated with splicing (data not shown).

The parents for which a DNA sample was available were found to be heterozygous for the respective mutations. None of the changes described were found in the controls.

Subcellular localization of the mutant MFSD8 proteins

Immunofluorescence analysis was performed as previously described (Siintola et al., 2007), to evaluate the impact of the novel CLN7/MFSD8 missense mutations on trafficking of the mutant MFSD8 proteins. The analysis showed colocalization of all the mutant proteins with the lysosomal marker LAMP1 (data not shown).

Clinical findings in CLN7/MFSD8 mutation positive patients

Clinical data were collected from the 25 patients found to have novel mutations in CLN7/MFSD8 (Table 2 and Fig. 2). Of these, 24 presented with a late-infantile onset phenotype with a mean age of onset of 3.3 years (range of 1.5–5 years). The presenting symptom in most patients was either developmental regression (10/24; 42%) or seizures (9/24; 38%). Progression was rapid with mental and motor regression, speech impairment and seizures developing to the majority of patients early in disease course (Fig. 2). Visual failure had developed to 90% and ataxia and myoclonus to 85% of patients. The mean age of death in the seven deceased patients was 11.5 years (range 6.5–18 years).

Figure 2

Schematic representation of the disease course in 24 CLN7/MFSD8 mutation positive patients with vLINCL. Each bar represents one symptom/finding, the length of the bar displaying the age range of onset. The black vertical lines on the bars show the mean age of onset of individual symptoms/findings. The age is given on the x axis in years. N = number of patients with the symptom/total number of patients for which information was available.

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Table 2

Clinical information of the CLN7/MFSD8 mutation positive patients identified in this study

Patient codeGenderAge of onsetAge of deathPresenting symptomMental regressionSpeech delay/ regressionMotor impairmentVisual failureAtaxiaSeizuresMyoclonousChairboundPathology (tissue)Mutation
270PaM2.5y13Regression+ (2.5y)+ (3−3.5y)+ (<4y)+ (retinopathy 4y)+ (3−3.5y)+ (3y10m)+ (7y)+ (6y)FP, CL (rectal biopsy)p.Arg35X
PPF2y9mAtaxia+ (3y)+ (3−3.5y)+ (3y)+ (3-3.5y)+ (3y)+ (3.5y)+ (4.5y)+ (5y)FP, GRODs, CL (lymphocytes)p.Arg139His
475PaM11yVisual failure+ (30y)+ (36y)+ (24y)+ (11y)+ (28y)+ (25y)No+ (39y)NAp.Thr156_Ala157delins Thr156_Pro157
397PaM4y16ySeizures+ (3y)+ (3y)+ (4y)+ (4.5y)+ (4.5y)+ (4y)+ (4y)NAFP, CL (muscle biopsy)p.Met209IlefsX3
N3403M5ySpecch delay, motor clumsinessNo+ (5y)+ (5y)NoNo+ (5y)+ (5y)+ (7y)NASplicing defect (c.863+1G>C)
N3703M3.5ySpeech delay, falling while walking+ (3.5y)+ (3.5y)NoNoNo+ (NA)NoNoNAp.Thr294Lys
N3704M4yDifficulty in walking and speaking+ (4y)+ (4y)+ (4y)+ (7y)NoNoNo+ (7y)NAp.Thr294Lys
447PaF4yNADevelopmental regression+ (4y)+ (4y)+ (4y)NA+ (4y)+ (4y)+ (4y)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
367PaF2.5yNASeizures, ataxia, speech regression+ (3y)+ (2.5y)+ (3y)+ (3y)+ (2.5y)+ (2.5y)+ (2.5y)+ (7y)RL/FP complex, CL (skin biopsy)p.Thr294Lys
367PbM3.5yNAAtaxia, atonic seizures+ (3.5y)+ (4y)+ (3.5y)+ (>5y)+ (3.5y)+ (3.5y)+ (4y)+ (8y)RL/FP complex, CL (skin biopsy)p.Thr294Lys
380PaF2.5yNASeizures+ (3y)+ (3y)+ (3y)NA+ (3y)+ (2.5y)NA+ (5y)RL/FP complex, CL (skin biopsy)p.Thr294Lys
427PaM4yNADevelopmental regression, visual failure+ (4y)+ (4y)+ (4y)+ (4y)+ (4y)+ (4.5y)+ (4.5y)+ (4y)RL/FP complex, CL (skin biopsy)p. Thr294Lys
466PaF3.5-4y6.5yDevelopmental regression+ (3.5y)+ (3.5y)+ (3.5y)+ (>4y)+ (3.5−4y)+ (4y)+ (NA)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
6M2y12yLoss of vision+ (2.5y)+ (2.5y)+ (2.5y)+ (2y)+ (2.5y)+ (NA)+ (NA)+ (7y)RL/FP complex, CL (skin biopsy)p.Thr294Lys
7F5yNASeizures, developmental regression+ (5y)+ (5y)+ (5y)+ (NA)+ (5y)+ (5y)NA+ (NA)RL/FP complex, CL (skin biopsy)p.Thr294Lys
8M4y18ySeizures, developmental regression+ (4y)+ (4y)+ (4y)+ (5y)+ (4y)+ (4y)+ (6y)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
 9M4.5yNADevelopmental regression, visual failure+ (4.5y)+ (4.5y)+ (4.5y)+ (4.5y)+ (4.5y)+ (4.5y)+ (5.5y)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
10M1.5yNADevelopmental regression+ (1.5y)+ (1.5y)+ (1.5y)+ (NA)+ (1.5y)+ (2.5y)+ (2.5y)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
12F2y7ySeizures+ (NA)+ (NA)+ (NA)NANA+ (2y)NANARL/FP complex, CL (skin biopsy)p.Thr294Lys
14M2yNADevelopmental regression+ (2y)+ (2y)+ (2y)NA+ (2.5y)NANANARL/FP complex, CL (skin biopsy)p.Thr294Lys
15M2y8yDevelopmental regression+ (2y)+ (2y)+ (2y)+ (2.5y)+ (2y)+ (4y)NA+ (4y)RL/FP complex, CL (skin biopsy)p.Thr294Lys
18F3yMotor impairment/speech regression+ (3y)+ (3y)+ (3y)+ (6y)No+ (5y)No+ (3y)NAp.Thr294Lys
16F4yNASeizures+ (4.5y)+ (4.5y)+ (4.5y)+ (retinopathy 4.5y)+ (4y)+ (3.5y)+ (3.5y)NARL/FP complex, CL (skin biopsy)p.Thr294Lys
11M3.5yNASeizures+ (4y)+ (4y)+ (4y)+ (retinopathy 5y)+ (4y)+ (3.5y)+ (3.5y)NARL/FP complex, CL (skin biopsy)Splicing defect (c.754+2T>A and c.1103- 2delA)
474PaM4.5NAMotor impairment+ (4.5y)+ (4.5y)+ (4.5y)+ (4.5y)NANA+ (4.5y)NAFP, RL, CL (skin biopsy)p. Arg465Trp
  • Manifestation of a particular symptom from each patient is indicated with a plus (+). The age at which the symptom presented is also given in parenthesis.

  • F = female; M = male; m = months; y = years; NA = not available.

Patient 475Pa presented with visual failure at 11 years of age and had a protracted disease course. Motor impairment and seizures developed at 24 and 25 years of age, respectively, followed by ataxia at the age of 28. Mental and speech regression were noticed at 30 and 36 years of age. At the age of 39, patient became wheelchair-bound. Today, patient 475Pa is 43-years old.

An ultrastructural examination of the storage material had been performed for 20 patients from various tissue samples (Table 2). The findings from these biopsies were reported by different pathologists. Electron microscopical (EM) examination revealed a complex of fingerprint (FP) profiles and rectilinear (RL) inclusions, occasionally associated with curvilinear (CL) profiles, in the majority of patients. The CLs were never the predominant finding, which would lead to suspicion of CLN2.

A brain computer tomography or magnetic resonance imaging (MRI) scan had been performed in 20/25 patients that were at different stages of disease progression. These revealed varying degrees of cerebral and cerebellar atrophy. Cerebral white matter signal changes especially on the periventricullar regions were reported in several patients on MRI. In addition, electroretinogram (ERG) findings were abnormal in the five patients for which ERG data were available.

Mutations in other NCL genes

Turkish patients (n = 45) found in haplotype analysis (see above) to be homozygous for other NCL loci were screened for mutations in the respective genes. A Roma patient from the former Czechoslovakia without a CLN7/MFSD8 mutation was also screened for CLN5 and CLN8 mutations. Three novel mutations in CLN2/TPP1 and one novel mutation in each of the CLN1/PPT1, CLN5, CLN6 and CLN8 genes were identified (Table 3). None of these changes were found in the controls. In addition, previously reported mutations for CLN2/TPP1 (c.622C>T/p.Arg208X), CLN6 (c.662A>G/p.Tyr221Cys; c.794_796delCCT/p.Ser265del) and CLN8 (c.473A>G/p.Tyr158Cys; c.709G>A/p.Gly237Arg) (NCL Mutation Database, http://www.ucl.ac.uk/ncl/) were identified. No mutations in the CLN3 gene were found in patient N0803 that showed homozygosity over CLN3.

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Table 3

Novel mutations in CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8

GeneNucleotide changePredicted amino acid change/ consequenceLocationPatient codeNumber of families with the mutationCountry of origin
CLN1/PPT1c.114G>Tp.Trp37CysExon 1SFT1Turkey
CLN2/TPP1c.17+1G>CSplicing defectIntron 1N44031Turkey
c.1204G>Tp.Glu420XExon 10N19031Turkey
c.1444G>Cp.Gly482ArgExon 12N21031Turkey
CLN5c.1026C>Ap.Tyr342XExon 4370Pa1Roma from the former Czechoslovakia
CLN6c.476C>Tp.Pro159LeuExon 4N14031Turkey
CLN8c.470A>Gp.His157ArgExon 2N30031Turkey


We here describe eight novel mutations, and a novel sequence variant, in the recently identified CLN7/MFSD8 gene (Table 1). These findings raise the total number of CLN7/MFSD8 mutations to 14, with the mutations scattered throughout the gene (Fig. 3). CLN7/MFSD8 was previously reported to underlie vLINCL in a subset of mainly Turkish patients (Siintola et al., 2007). We show that CLN7/MFSD8-associated defects are not restricted to the Turkish population, but instead are geographically more widespread than originally anticipated. Our findings emphasize the importance of including CLN7/MFSD8 in a diagnostic scheme of vLINCL patients.

Figure 3

Schematic representation of the CLN7/MFSD8 gene shows the relative positions of the mutations. The exons are shown in scale as numbered cylinders. The untranslated fragments are depicted with darker colour. The introns are shown as lines and are not in scale. The previously reported mutations (Siintola et al., 2007) are depicted in the upper part of each exon, and the new ones described here in the lower parts.

The new CLN7/MFSD8 mutations identified here co-segregate with the disease phenotype in the respective families and were absent in 200 control chromosomes. High conservation of the affected amino acids, together with the in silico predicted deleterious effect on the protein function, support the pathogenic nature of the four amino acid changing mutations. Further, immunofluorescence analysis of the mutant proteins suggests disturbed functional properties, rather than altered subcellular localization, as the primary consequence of these mutations. Both the nonsense mutation p.Arg35X and the deletion mutation p.Met209IlefsX3, introduce premature stop codons, the mRNAs most probably being degraded through nonsense-mediated decay (Hentze and Kulozik, 1999). Finally, the nucleotide changes c.863 + 1G>C and c.1103-2delA are likely to affect the donor splice-site of intron 9 and the acceptor splice-site of intron 11, respectively. The outcome of these mutations could not be evaluated because RNA samples from the respective patients were unavailable. Nevertheless, both changes affect invariant nucleotides at the splice-sites and thus most likely alter the splicing pattern, producing abnormal transcripts (Cartegni et al., 2002).

A ninth sequence variant, c.63-4delC, was identified in a patient that was previously found to be heterozygous for a paternally derived nucleotide change, c.1738G>A (p.Val580Met), in the CLCN6 gene (Poet et al., 2006). Interestingly, the c.63-4delC alteration was also inherited from the father, thus excluding digenic parental inheritance. The c.63-4delC alteration was not present in 200 control chromosomes and RT-PCR analysis showed no evidence for altered splicing in the patient sample. Thus, it remains open whether c.63-4delC is a disease-associated mutation or a rare non-pathogenic polymorphism.

Previously, Roma patients from the former Czechoslovakia have clinically been considered to have early juvenile CLN6 variant (Elleder et al., 1997). While linkage to the CLN6 locus supported this diagnosis (Sharp et al., 1999), no CLN6 mutations were identified (Sharp et al., 2003). We here demonstrate that, contrary to the previous linkage data, the majority of the Roma NCL patients (n = 14/15) are homozygous for the missense mutation c.881C>A (p.Thr294Lys) in CLN7/MFSD8. Haplotype analysis over the CLN7 locus in these patients was consistent with the existence of a common founder effect (data not shown). One Roma patient was found to be homozygous for a novel truncating p.Tyr342X mutation in CLN5, which is most likely a private mutation in the family. Identification of a founder mutation in the majority of the Roma patients is of primary importance for the development of a diagnostic test for this population.

With the exception of one, all mutations identified in CLN7/MFSD8 to date are associated with a similar clinical course of late-infantile onset that is predicted to be equivalent to complete loss of gene function. The age of onset in the CLN7/MFSD8 mutation positive patients with a late-infantile onset ranges from 1.5 to 5 years (this study; Topcu et al., 2004; Siintola et al., 2007) and thus overlaps with that of the other late-infantile forms (Mole et al., 2005). Moreover, the clinical presentation and disease course in CLN7 are essentially indistinguishable from that in other LINCL forms. Finally, the ultrastructural pattern of the storage material in CLN7 patients, consisting of RL/FP complex occasionally associated with CL inclusions, closely resembles EM findings in the other vLINCL forms, the classical late-infantile CLN2 form being distinguished from the other forms by the invariant presence of CL bodies (Haltia, 2003). In the group of vLINCL, molecular genetics methods are thus required to make the distinction between the clinically and ultrastructurally similar entities. In the case of CLN7/MFSD8 sequencing of the gene remains the most practical alternative for diagnostics, as the majority of patients seem to have private mutations.

Contrary to the vLINCL group of CLN7/MFSD8 mutation positive patients, homozygosity for a mutation resulting in substitution of a neutral non-polar alanine at position 157 by another neutral non-polar proline was associated with a later onset, and more protracted disease course in patient 475Pa. The later onset with visual failure as the presenting symptom implies that in differential diagnosis of Batten disease or juvenile onset NCL patients that remain negative for CLN3 mutations, CLN7 should be considered as an alternative diagnosis. Similar to many other NCL genes (Mole et al., 2005) mutations in CLN7/MFSD8 may underlie atypical, later onset and milder phenotypes and should thus be considered a candidate in NCLs beyond late-infantile onset.

Turkish vLINCL was originally thought to represent a distinct clinical and genetic entity (Wheeler et al., 1999). It is now evident that this group of patients, despite being clinically relatively uniform (Topcu et al., 2004), is genetically heterogeneous with mutations described in three vLINCL genes (present study; Ranta et al., 2004; Siintola et al., 2005; Siintola et al., 2007). Lack of comprehensive clinical evaluation including measurement of PPT1 and TPP1 activities, as well as ultrastructural analysis of the storage material, may have lead to incorrect clinical diagnosis of vLINCL in five patients in whom we identified homozygous mutations in either CLN1 or CLN2. In this study, all known human NCL loci were excluded in Turkish vLINCL patients from 35 families. Thus, knowledge of the genetic spectrum underlying vLINCL is still far from complete. Our previous genome-wide linkage study provided evidence for the existence of at least three more genes underlying vLINCL in five different families (Siintola et al., 2007). Ensuring an accurate diagnosis in patients not linked to any of the known human NCL loci, in combination with genome-wide homozygosity mapping in these remaining patients, should lead to the identification of these novel NCL genes.

Supplementary material

Supplementary material is available at Brain online.


We thank the families that participated in this study, as well as Marianne Rohrbach, Stacey Hewson, Eva Kostalova, Gul Serdaroglu, Larisa Stolnaja, Claudia Kitzmüller, Ahmed Mohamed and Teija-Tuulia Toivonen for their contribution. This study was supported by the Center of Excellence in Complex Disease Genetics of the Academy of Finland, the Folkhälsan Research Foundation, the Institute of Inherited Metabolic Disorders support (project MSM 0021620806), the Ministry of Health of the Czech Republic grant (NR/8351-3), the Wellcome Trust (054606) and the Batten Disease Support and Research Association. M.K. is fellow of the Helsinki Biomedical Graduate School. B.A.M. holds a Canada Research Chair in Pediatric Neurogenetics.


  • Abbreviations:
    endoplasmic reticulum
    neuronal ceroid lipofuscinoses
    variant late-infantile onset NCL


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