Background
The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited lysosomal storage disorder causing severe neurodegeneration due to neuronal loss (brain and retina), and accumulation of lipopigments in many cell types, including neurons. The NCLs incidence rate worldwide is 1 to 8 in 100,000 live births [
1].
The NCLs share common clinical presentations like epilepsy, loss of motor and cognitive function, visual impairment, and premature death [
2]. Though usually observed in childhood, the age of onset of the disease varies. Considering this, NCLs were initially classified into four groups – infantile (INCL-Haltia-Santavuori disease), late infantile (LINCL-Jansky-Bielschowsky disease), juvenile (Batten-Spielmeyer-Vogt disease) and adult (Kufs disease) [
3]. Eventually, allelic heterogeneity in NCLs was identified due to an advancement in biochemical and genetic techniques and hence a new approach of molecular classification and diagnostic algorithms was designed [
4,
5].
Until now, 14 types of NCL are identified (NCL1-NCL14) however the most commonly observed form are NCL1, NCL2, and NCL3 [
6]. These NCLs subtypes are autosomal recessively inherited except for NCL4B, inherited in an autosomal dominant form [
7]. An exception to the above inheritance pattern is a uniparental disomy case in NCL which occurred due to complete isodisomy of chromosome 8, leading to homozygosity of a maternally-inherited deletion in NCL8 [
8]. Until May 2015, total 515 changes in 13 human genes have been reported in NCLs [
9].
NCL1 (OMIM#256730) representing the early infantile disease, results due to a mutation in
PPT1 (palmitoyl-protein thioesterase 1; OMIM*600722) gene located at 1p34.2. The gene codes for a lysosomal enzyme called palmitoyl-protein thioesterase 1 (PPT1) whose function is to remove fatty acids attached in thioester linkages to cysteine residues in the protein. The downstream effect of PPT1 deficiency involves deregulated cellular processes like vesicular trafficking, synaptic function, lipid metabolism, neural specification, and axon connectivity [
10]. In addition, a study by Lyly et al. established that an alteration in cholesterol metabolism and ectopic F1-ATP synthase resulted due to PPT1 deficiency [
11]. According to the NCL mutation and patients’ database 76 changes have been reported in
PPT1 gene [
9]. Amongst these, the mutation p.Arg122Trp has a founder effect in Finnish population with NCL1 [
12]. This mutation causes a defect in the transport of the PPT1 from the endoplasmic reticulum to lysosomes [
12,
13]. The mutation p.Thr75Pro and p.Leu10Ter have a founder effect in Scotland [
14].
NCL2 (OMIM#204500), representing the late infantile disease, results due to a defect in
TPP1 (tripeptidyl peptidase I; OMIM*607998) gene at the locus 11p15.4. This gene encodes the instruction for making the lysosomal enzyme called tripeptidyl peptidase 1 (TPP1). TPP1 deficiency results in accumulation of ceroid lipofuscin, an autofluorescent storage material, in cell’s lysosomes. Total 140 disease-causing mutations are in the
TPP1 gene of the patients with NCL2 [
9]. The most common
TPP1 gene mutations are c.509-1G > C and p.Arg208Ter [
14]. The mutation p.Gly284Val seems to be predominant in Canada suggesting a possible founder effect [
14].
The genetics of NCL1 and NCL2 remain unknown in India. Hence, the aim of the present study is to identify the molecular spectrum and common molecular marker of these diseases in Indian patients. The study also aims to support the correlation between the null or reduced enzyme activity and the mutations causing disease and clinical phenotype in NCL1 and NCL2 patients.
Discussion
Data presented here is the first study from India demonstrating the mutation spectrum of Batten disease (NCL1 and NCL2) in a large cohort. The given study reveals 34 cases of NCLs (12 with NCL1, and 22 with NCL2) with maximum NCL2 cases. Similarly, in a study by Santorelli et al., the highest numbers of cases confirmed the NCL2 (24%) [
29].
The patients’ clinical appearance like seizures, myoclonic jerk, visual impairment, and neuroimaging examination showing cerebral atrophy and cerebellar atrophy were in concordance with the previously established phenotypes in NCL patients [
3]. The mutations identified in this study resulted in a broader spectrum of clinical presentation and hence hampered the genotype-phenotype correlation in NCL patients. Such clinical presentations due to
PPT1 and
TPP1 gene mutations are also observed in the orthologous species. For instance, a study by Sanders et al. identified a homozygous mutation c.736_737insC in exon 8 of
PPT1 gene in a canine presenting NCL-like signs including, visual impairment, disorientation, behavioral changes, lack of PPT1 activity in the brain, and accumulation of autofluorescent lysosomal inclusions with the granular osmiophilic deposit in neurons [
30]. Also, a study by Mahmood et al. established that a homozygous
TPP1 gene mutation in a zebrafish results in the progressive early onset of neurodegenerative phenotypes, small retina, accumulation of subunit c of mitochondrial ATP-synthase, and localized apoptotic cells death in the retina, optic tectum, and cerebellum [
31].
Several country-specific mutations are reported in NCL1 and NCL2. The most common NCL1 mutation identified in Finland is p.Arg122Trp in
PPT1 gene, which accounts for 98% Finish variants [
12]. A study by Das et al. revealed two common mutations, p.Arg151Ter and p.Thr75Pro, in
PPT1 gene of American NCL1 patients [
32]. The absence of these common mutations in the present study of Indian patients suggests the molecular heterogeneity of NCL1 in India. In this study, a novel variant c.713C > T (p.Pro238Leu) was identified in the
PPT1 gene of four unrelated NCL1 positive families (44%) from the southern part of India. This suggests its possible founder effect in the Indian origin settlers. However, a detailed study in larger cohorts is essential.
In case of NCL2, two common mutations p.Arg208Ter and c.509-1G > C (as per old nomenclature T523-1G > C) accounting for approximately 60% of all identified
TPP1 mutant alleles worldwide and at least one of these mutation can be identified in more than 75% of patients [
33,
34]. In the present study, one patient with NCL2 was identified with a homozygous p.Arg208Ter mutation but the variant c.509-1G > C was not observed in our cohort which indicate its uncommon occurrence in Indian NCL2 patients. The NCL2 country-specific mutation includes p.Gly284Val in Canada and p.Asp276Val in Argentina [
14,
35]. In the present study, a known pathogenic mutation p.Arg206Cys was observed most commonly in the unrelated NCL2 patients (26%) suggesting its possible founder effect.
However, in the given study, the genetic diagnosis of about 17% of patients remained ambiguous. A similar percentage was also observed in previously published data were around 10% of patients were without any genetic identification [
29]. This suggests that the deep intronic variants, large deletion or duplication in the NCL genes might also play a role in disease occurrence. In addition, as suggested by Santorelli et al., studying large informative families might identify new NCL genes and help in understanding NCLs molecular pathology [
29].
A study by Das et al. established that a reduction in PPT1 and TPP1 enzyme activity ranges from 0 to 2.5% [
33]. In the given study also, the reduction in these enzymes activity was from 0 to 2.8%. Based on these biochemical observations, the therapeutic approaches are tested to regain the enzyme activity. For instance, a study in a canine model with TPP1-deficiency revealed that the administration of a recombinant adeno-associated virus (rAAV) expressing canine TPP1 in the ependyma resulted in elevation of TPP1 expression leading to delay in clinical presentation and extension of life span [
36]. In addition, studies to diminish the clinical phenotypes of NCL have been directed. Tracy et al. reported an alternative approach of using stem cell based delivery of therapeutic components to the retina, as the systemic administration would be ineffective [
37]. This study reported the inhibition of the retinal degeneration in the canine model after a single intravitreal administration of autologous bone marrow-derived stem cells transduced with a TPP1 expression construct [
37].
Acknowledgments
We show our gratitude to the proband and their families for their support and without whose consent this study would not have been possible. We also appreciate the suggestions from Task Force members (Dr. Ratna Puri, Dr. Seema Kapoor, Dr. Ashwin Dalal, Dr. Subha Phadake, Dr. Girisha Katta, Dr. V Shankar) for referring patients at our institute. We also acknowledge Department of Health Research, Indian Council of Medical Research (ICMR), Government of India as a part of Multicentric National Task Force on Lysosomal storage disorders (Project No: GIA/31(ii)/2014-DHR) for supporting this work.