Clinical and genetic characterization of neuronal ceroid lipofuscinoses (NCLs) in 29 Iranian patients: identification of 11 novel mutations

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative lysosomal storage diseases which are considered among the most frequent causes of dementia in childhood worldwide This study aimed to identify the gene variants, molecular etiologies, and clinical features in 23 unrelated Iranian families with NCL. In total, 29 patients with neuronal ceroid lipofuscinoses (NCLs), diagnosed based on clinical manifestations, MRI neuroimaging, and electroencephalography (EEG), were recruited for this study. Through whole-exome sequencing (WES), functional prediction, Sanger sequencing, and segregation analysis, we found that 12 patients (41.3%) with mutations in the CLN6 gene, 7 patients (24%) with the TPP1 (CLN2) gene variants, and 4 patients (13.7%) with mutations in the MFSD8 (CLN7) gene. Also, mutations in each of the CLN3 and CLN5 genes were detected in 2 cases and mutations of each PPT1 (CLN1) and CLN8 gene were observed in only 1 separate patient. We identified 18 different mutations, 11 (61%) of which are novel, never have been reported before, and the others have been previously described. The gene variants identified in this study expand the number of published clinical cases and the variant frequency spectrum of the neuronal ceroid lipofuscinoses (NCLs) genes; moreover, the identification of these variants supplies foundational clues for future NCL diagnosis and therapy.


Introduction
Neuronal ceroid lipofuscinoses (NCLs) are complex group disorders of inherited severe neurodegenerative lysosomal storage diseases that usually onset is most often in childhood, sometimes in adulthood (Zeman and Dyken 1969;Mole et al. 2005;Mole et al. 2011). This disease has been identified as one of the most frequent childhood-onset neurodegenerative disorders. The prevalence of NCLs has been estimated at 1:1,000,000 to 1:14,000 worldwide; however, this far varies between geographical regions and different races (Williams and Mole 2012;Sleat et al. 2016;Simonati and Williams 2022). Fourteen NCL types (CLN1 to CLN14) have been described to date (Mole and Cotman 2015;Mink et al. 2013;Gardner and Mole 2021;Warrier et al. 2013). Almost all of them are inherited in an autosomal recessive manner except one adult-onset form caused by mutations in DNAJC5/CLN4 that have been reported in autosomal dominant inheritance (Mole and Cotman 2015;Nosková et al. 2011). NCL has two major hallmarks: first accumulation of autofluorescent storage material (ceroid) in the lysosome in the neurons and other cells and second neurodegeneration that affects CNS neurons such as ganglionic neuronal cells of the retina (Zeman and Dyken 1969;Seehafer and Pearce 2006). From clinical perspective, NCL is mainly presented by epileptic seizures, mental and motor deterioration, visual impairment leading to blindness, progressive ataxia, dementia, and reduced life expectancy (Mole et al. 2011;Haltia 2003;Haltia and Goebel 2013). Classically, NCLaffected individuals have been classified into four categories (infantile, late infantile, juvenile, and adult) which have been mainly defined regarding the clinical onset of symptoms; however, some patients cannot be easily included in a specific group because of the significant variation in the age of onset and disease progression. Therefore, it is preferred to classify NCL based on the genetic etiology of known genes. now, with the advent of molecular diagnosis and identification of causative genes, a novel NCL classification has been suggested taking into consideration the gene and mutation first and then with clinical phenotype, biochemical aspect, ultrastructural features pathological features (Mole et al. 2005;Tomson et al. 2015;Kousi et al. 2012;Kaminiów et al. 2022). Nowadays, 13 genes are related to NCLs disease, including PPT1, TPP1, CLN3, DNAJC5, CLN5, CLN6, MFSD8, CLN8, CSTD, GRN, ATP13A2, CTSF and KCTD7 are detected (Williams and Mole 2012;Mink et al. 2013;Brudvig and Weimer 2022). Based on the UCL database, more than 430 NCL etiological mutations have been reported in these genes (https:// www. ucl. ac. uk/ ncl-disea se). Hence, the increasing implementation of next-generation sequencing panels and exome sequencing as essential diagnostic tools will lead to more accurate and faster diagnoses in patients with NCL, including those that differ from the known phenotypes usually due to so-called milder mutations (Williams and Mole 2012;Srivastava et al. 2014).
We enrolled 29 patients from 23 separated families with Batten disease symptoms in this study. All probands were evaluated using NGS technology.We found that 12 patients (41.3%) with mutations in the CLN6 gene, 7 patients (24%) with the TPP1 (CLN2) gene variants, and 4 patients (13.7%) with mutations in the MFSD8 (CLN7) gene. Also, mutations in each of the CLN3 and CLN5 genes were detected in 2 cases (6.8%) and mutations of each PPT1 (CLN1) and CLN8 gene were observed in only 1 separate patient (3.4%). We identified 18 different mutations, 11 (61%) of which are novel, never have been reported before, and the others have been previously described.

Ethical compliance
The study was approved by the ethics committee of the Institutional Review Board (IRB), with the approval code of IR.SBMU.MSP.REC.1400. 250 and carried out following the tenets of the Declaration of Helsinki (World Medical Association 2001). Written informed consent was obtained from the patient's parents for the publication of this report, data, and any accompanying images.

Patients
Patients with initial symptoms of seizures, myoclonus, cognitive and motor function decline, ataxia, behavioral problems, visual and speech impairment were investigated. Families found to be affected by environmental and nongenetic factors were excluded from the study. Clinical data provided by the referring Child neurologist were annotated in conformity with the Human Phenotype Ontology (HPO) nomenclature (Köhler et al. 2021).
After choosing the candidate families, precise genetic counseling was accomplished with each family, any information about them, including the family history of diseases, was carefully recorded, and finally, a family pedigree was drawn. Complementary medical and clinical examinations such as MRI neuroimaging, electroencephalography (EEG), and eye examination were performed for all probands and all data were recorded.
The present study included 23 families consisting of 29 affected individuals together with their healthy family members, suspected of NCL disease. Five families presented more than one affected patient one of them is a twin brother and the remaining 16 families presented with one affected case. All of them were born into families with healthy parents and were normal at birth (Fig. 1).

Exome capture, sequencing, and bioinformatics analysis
Whole blood samples were taken from patients and their available family members. Genomic DNA was extracted using DNA extraction standard salting out protocols.
Whole-exome sequencing (WES) was carried out in all 23 probands using a custom-designed Nimblegen chip-capturing array. Nearly 60 Mb of the targeted region on consensus coding sequences enriched from fragmented genomic DNA of the proband by around 758,086 probes designed for the human genome (Agilent SureSelectXT2 V7 exome). Then sequencing was performed on the Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA). Paired-end sequence reads were aligned to the human reference genome (UCSC hg19/GRCh37) using Burrows-Wheeler Aligner (BWA). Variant calling was run using SAMTools and Genome Analysis Toolkit (GATK v 3.7) (Li and Durbin 2010;Li et al. 2009;McKenna et al. 2010). Moreover, ANNOVAR software annotated and filtered the variants.

Variant confirmation and co-segregation analysis
To confirm the variant in the probands, their parents, and also available siblings, bi-directional Sanger sequencing was applied. Referenced and designed primer sequences of the NCL genes were extracted from the National Center for Biotechnology Information (NCBI) database. Primers (F and R primers for mutation PCR) designed by Primer3 (http:// prime r3. ut. ee/) (available upon request), any possible secondary structures of the designed primers were predicted using Gene Runner software version 3.01. Then, we conducted the polymerase chain reaction (PCR) under standard conditions. The samples were sequenced using ABI Prism3130 Genetic Analyzer (Applied Biosystems), and the obtained results were analyzed using Chromas 2.5 (chromas. software.informer.com) software.

In silico evaluation of the pathogenicity of candidate mutations
To predict the pathogenicity of the variant, we used different bioinformatics software and tools. The raw reads were filtered as clean reads and then aligned to the GRCh37 (hg19) human reference sequence. At first, variants were preferentially selected for further analysis and validation with their minor allele frequency (MAF) < 0.01 in the ExAc browser (exac.broadinstitute.org), 1000 genome databases (http:// brows er. 1000g enome. org), Iranome (http:// www. irano me. ir) and gnomAD (https:// gnomad. broad insti tute. org) database. Also, the variants had to be located in exons, and introns regions that affected RNA splicing or regulatory elements.
Then, single-nucleotide polymorphism (SNP) and short indel candidates were identified, and the data were extracted from the Ensemble.org, dbSNP (http:// www. ncbi. hlm. nih. gov/ SNP), Clinvar, Franklin, VarSome, OMIM database, HGMD (http:// www. hgmd. cf. ac. uk) and recently published articles in PubMed. The SIFT utilities were used to forecast the change in protein structure. Conservation of each amino acid change was calculated using PhyloP2 and Mutation-Taster (http:// www. mutat ionta ster. org/) algorithms were used to predict the effects of variants on protein function.
All novel variants were named according to the guidelines of the Human Genome Variation Society (http:// www. hgvs. org/) and were described and classified based on the standard guidelines of ACMG/Association for Molecular Pathology, and are scored based on the evidence and strength of each criterion as described in the ACMG guidelines.

Genotype-phenotype associations
The novel candidate variant or previously reported mutation to cause NCL disease was scrutinized and adapted to the clinical symptoms of each patient. They were also determined using several databases such as Human Gene Mutation Database, Leiden Open Variation Database, PubMed (https:// www. ncbi. nlm. nih. gov/ pubmed/), and Online Mendelian Inheritance in Man (https:// omim. org/).

Clinical data
This study includes 23 families with 29 patients suffering from NCL disease. We should emphasize that the clinical diagnosis of all the NCL cases is in line with the diagnostic criteria that have already been approved for this disease including seizure, myoclonic epilepsy, regression, ataxia, visual impairment, and behavioral problems. Clinical symptoms were presented for 29 cases and cataloged based on Human Phenotype Ontology (HPO) terms are summarized in Table 1 (Köhler et al. 2021;Trujillano et al. 2017).
The patients were followed over months to years (6 months-4 years) in a neurology and genetic outpatient clinic in our teams. During this time, five patients passed away due to complications caused by the disease (P2, P3, P5, P18 and P22).
In this study, eighteen homozygous variants in 7 different genes were detected in 23 families studied. These variations included seven previously reported and eleven apparently novel variants. A summary of detected mutations is provided in Table 2.

Patients with mutations in the CLN6 gene
Mutation in the CLN6 (NM_017882.3) gene cause Ceroid lipofuscinosis, neuronal, 6A (OMIM#601780) disorder with a variable age of onset from 1.5 to 5 years of life after normal early development (Sharp et al. 2003) and Ceroid lipofuscinosis, neuronal, 6B (Kufs type OMIM# 606725) form of 'Kufs disease,' which refers in general to adult-onset neuronal ceroid lipofuscinosis without retinal involvement (Arsov et al. 2011), with autosomal recessive manner.
Family 3 has three affected children with very similar symptoms and natural history. P4, the proband, was homozygous for a missense mutation in exon 6 of the CLN6:c.662A>C. This Likely Pathogenic mutation; has been reported previously. Sanger sequencing confirmed this mutation in both P5 and P6 patients. F5 expired at age 10 due to respiratory failure.
NGS analysis individually in P12 and P17 samples revealed a novel VUS (PP3, PM2, PP2) missense mutation in exon 7 (c.758T>C, p.L253P), of the CLN6 gene. This mutation affects a highly conserved residue, this change presumably disrupts the function of the protein.
We identified another novel variant in the P18 patient. This missense Likely Pathogenic variant is located in exon 7 of the CLN6 (c.710C>T, p.Thr237Ile) and may damage the protein structure and function according to the prediction tools.
In P19 proband, we found out a missense variant in exon 4, CLN6: c.407G>A. This Pathogenic mutation has been reported before and showed that it affects p.Arg136His; also in index P21, we reported a Likely Pathogenic mutation in exon 3, CLN6:c.252C>G. This nonsense mutation affects protein features (might be) and introduces an NMD.
Finally, in patient P28, we detected one more novel splice-site intronic mutation in intron 3 of the CLN6 (c.486+5G>A) gene. This variant was classified as a variant of uncertain significance (VUS), but it showed some pathogenic indicatives based on in silico prediction programs, including the Human Splicing Finder and MaxEntScan.
This VUS variant splice-site change was predicted by in silico analysis to affect the downstream domain in the protein.

Patients with mutations in the TPP1 gene
Ceroid lipofuscinosis, neuronal, 2 (OMIM#204500) underlies the pathogenesis of late-infantile NCL (LINCL) caused by a mutation in the TPP1 (NM_000391.4) gene (Schulz et al. 2018). We identified homozygous disease-causing mutations in TPP1 gene in 5 studied families (F2, F5, F9, F10 and F21) ( Table 2). In these seven patients, the median age of onset was 2.4 (late-infantile) years. five cases (P2, P3, P8, P9, P13, P16 and P27) showed the symptom of the disease with myoclonus seizure; and the rest of them manifested the disease by ataxia. All patients had myoclonus seizure, mental and developmental regression, ataxia, severe speech defect, and visual impairment, patient P2 was blind. Cerebellar atrophy in P2, P3, P8, P9 and P14, and Cerebral atrophy in P13 and P27 patients were detected in their brain MRI.
In family (F2), indexes P2 and P3 correspond to two sisters who passed away, respectively, at 6.5 and 14 years old. The progression of the disease in P2 was much faster than in her sister. Unfortunately, both sisters died due to respiratory complications. WES was performed for P2 and showed a novel missense mutation in exon 10 of the TPP1 (c.1205A>G, p.E402G) gene. This Likely Pathogenic (PP3, PM2, PP2, PP5) mutation was never reported before. The presence of this variation in indexes P2 and P3 was validated by Sanger sequencing. Surprisingly, this mutation was also found by WES in another family (F5). This family has two (11 and 7 years old) brothers. In proband (P8) NGS analysis showed this likely pathogenic mutation. We checked this finding in his brother (P9) and found that he has the same variant.
WES in P13 showed a recurrent nonsense mutation exon 6 of the TPP1 (c.622C>T, p.R208X) gene. This mutation converts a highly conserved amino acid, Arg208, into a stop codon, a change that can affect the amino acid sequence and properties of the protein, and also induces NMD. We found another novel missense variant in P16, this mutation (c.1568A>G, p.H523R) is detected in exon 13 of the TPP1 gene and affecting the highly conserved His523 residue, and has not been reported previously. This variant, classified as VUS (PM2, PP3, PP2), was predicted by in silico analysis to have a detrimental effect on protein function.
The last mutation that we detected in TPP1 was in P27, which recently showed symptoms of the disease.This Pathogenic missense mutation (c.1551+1G>T)-affected splice site has been reported twice before.
Three families (F4, F17 and F19) had homozygous mutations in the MFSD8 gene (Table 2). All four (P7, P22, P24 and P25) patients presented a typical form of CLN7 and their symptoms were started at median age of 3.3 years old (late infantile) by ataxia (P7 and P22) and seizure (P24 and P25).
P7, P24 and P25 have refractory seizures and P22 had myoclonus seizures; he died at 13 due to disease complications. All of them had mental and developmental regression, ataxia, and blindness. speechless and cerebellar atrophy was observed in P7 and P22, while speech defect and cerebellum atrophy were detected in P24 and P25.
A pathogenic novel mutation in exon 7, MFSD8 (c.616C>T, p.Q206X) was detected with WES in two (P7 and P22) probands separately. It is the first time that this pathogenic (PVS1, PM2, PP5) mutation is reported. Both patients have almost the same course of the disease. Unlike other patients with CLN7, they did not have delayed psychomotor development but suffered from a sleep disorder, dysphagia, and bladder and bowel incontinence. This nonsense mutation converts Gln206 into a stop codon which can affect protein features and introduce an NMD, so this change entirely destroys the function and structure of MFSD8 protein.
Family 19 has a twin boy patient. WES was performed for P24 and manifested a novel missense variant (c.847T>G, p.F283V) in exon 9. This VUS (PM2) mutation affects a highly conserved residue (Phe283) and predicting this conversion should interrupt the protein function. The presence of this mutation in F25 was accredited by Sanger sequencing.

Patients with mutations in the CLN5 gene
Homozygous mutation in the CLN5 (NM_006493.4) gene is observed in Ceroid lipofuscinosis, neuronal, 5 (OMIM#256731) patients. In both cases from two families (F18 and F20) with CLN5 mutations (Table 2), the age of onset was around 7 years (juvenile).
Index P23 started the symptoms of the disease with ataxia and after that myoclonus seizure. The symptoms continued with continuous neurological and motor regression, speechlessness, and blindness. NGS showed that she has a novel missense mutation in exon 3 of NCL5 (c.476G>A, p.C208Y). This VUS (PM2, PP3, PM1) variant changes a highly conserved residue Cys208.Proband P26 showed the disease with a speech defect. Myoclonus seizure, ataxia, regression, and progressive visual loss were started. WES revealed another novel Likely Pathogenic mutation in exon 1 of the CLN5 (c.172A>T, p.K107X) gene. This stop-gain mutation shifted Lys107 into the termination codon, which can shorten the amino acid sequence, and protein features affected and also induced NMD.

Patients with mutations in the CLN3 gene
Ceroid lipofuscinosis, neuronal, 3 (OMIM#204200) juvenile NCL (JNCL) is caused by a homozygous mutation in the CLN3 (NM_001042432.2) gene. In this study, we described a family (F11) with two sisters that presented the disease at 8 years old (Late-juvenile) by myoclonus seizure ( Table 2). Both of them have regressing, ataxia, speech, and visual defects, but the elder sister P15 shows more severe and advanced symptoms of the disease. WES was performed for P15 and showed a novel truncating mutation deletion c.1274_1275delTC, in exon 10 of the CLN3. Although this frameshift mutation (p.L425Pfs*7) is classified as VUS (PM2, PVS1) according to the ACMG classification but causes truncated protein and NMD as predicted by prediction tools.
Also, Ceroid lipofuscinosis, neuronal, 8 (OMIM#600143) is caused by a homozygous mutation in the CLN8 (NM_018941.4) gene ( Table 2). The symptom of illness in P20 started with speech defect, then she showed ataxia and refractory myoclonus seizure, visual impairment, and bladder-bowel incontinence. Brain MRI indicated cerebellar signs. WES analysis in her sample revealed a novel pathogenic (PP5, PM2, PM5, PP3, PM1) homozygous missense mutation in exon 2 of the CLN6 (c.208C>T) gene. This mutation affects a highly conserved residue Arg70 and converts it to Cys.

Discussion
Neuronal ceroid lipofuscinoses (NCLs) are considered among the most frequent cause of dementia in childhood worldwide (Williams and Mole 2012). They are a group of genetically and clinically heterogeneous disorders that are determined by the accumulation of abnormal storage material NCL-specific lipopigments in neurons and the other cells in the body. Abnormal ceroid storage led to neuronal death in all gray regions of the brain. Damage to the white matter, rate of progress and extent of involvement vary and depend on the type and severity of disease progression (Simonati and Williams 2022;Haltia 2003;Santavuori 1988;Roine et al. 2019). The molecular mechanisms of linking the formation of endo-lysosomal storage and death of the neuronal cells have not so far been clearly manifested. The primary defect of lysosomal proteolytic activity is present in only three NCL forms, including CLN1/PPT1 (palmitoylprotein thioesterase 1), CLN2/TPP1 (tripeptidyl peptidase I) and CTSD/CLN10 [cathepsin D (CTSD)]. Also, we knew that DNAJC5 (CLN4) gene encodes a Hsc70 co-chaperone involved in exocytosis and endocytosis, whereas in the remaining NCLs, mostly encode membrane proteins with unknown functions (Mole 2004;Kollmann et al. 2013;Nelson 2007).
NCLs have a degenerative disease course and are characterized by myoclonic epilepsy, progressive movement and developmental regression, ataxia, different forms of visual impairment such as optic atrophy, retinal degeneration, retinopathy evolving into blindness, and variable cerebellar and cerebellum atrophy. These features lead to the disease being recognized as a disorder with a poor prognosis, significantly reducing sufferers' life expectancy. The age at disease onset ranges from birth to adulthood and the severity and type of symptoms are different depending on the subtype of NCLs (Mole et al. 2005;Nita et al. 2016). During the last decade, advances in understanding the underlying pathophysiology of NCLs, through the identification of new genes, mutations and learning about the pathways involved in the formation and development of the disease, have led to the synthesis of several helpful drugs. In April 2017, the FDA approved Ceroliponase alfa (Brineura) recombinant human tripeptidyl peptidase (TPP) 1 for the treatment of NCL2, an innovative treatment available for this rare disease (Lewis et al. 2020;Geraets et al. 2016;Markham 2017).The estimation of the prevalence of this disease varies from 2 to 4 in 1,000,000 in Western countries. In IRAN, a large country with an estimated population of over 85 million, we still do not have an exact estimate of the NCLs disorders prevalence. However, the high consanguinity marriage frequency in Iran might imply an expected high risk for inherited disorders, especially autosomal recessive traits (Cotman et al. 2013;Mukherjee et al. 2019).
The data presented here are the first study from Iran demonstrating the genetic and clinical data of NCL disease in a cohort. This study reveals 29 diagnostic cases (18 unrelated and 11 siblings) of NCLs. Diagnosis of NCLs was based primarily on clinical findings. The clinical manifestations of our cases such as seizure, myoclonus, progressive decline of cognitive and motor capacities, ataxia, progressive visual loss, speech defect, as well as complementary medical and clinical examinations such as MRI neuroimaging (showing cerebral atrophy and cerebellar atrophy), electroencephalography (EEG) and eye examination were in concordance with the previously affirmative phenotypes in NCL patients. As expected, cerebral and cerebellum atrophy and cerebellar signs were observed in our probands, the results of the EEG also suggested brain damages in the proband and the eye examinations showed different forms of visual impairment (from mild to severe and blindness).
All of our patients proved to carry a homozygous variant in the NCL's gene. Consanguinity was noted in 21 out of the 23 pedigrees (91.3%) (Fig. 1). This high rate represents high homogeneity because of the high consanguineous marriage rate among the Iranian population. We detected an identical novel variant in the PPT1 (c.1205A>G) gene in two completely separate families (F2 and F5), each with two affected children. Also, we presented a stop-gain mutation for the first time in the MFSD8 (c.616C>T) in two different families (F4 and F17). Also, another not reported variant in the CLN6 (c.758T>C) was found in this study in two unrelated families (F8 and F12).
Among our reported mutations, 10 (55.5%) were missense, 4 (22.2%) nonsense (stop gain), 2 (11.1%) splice site, 1 (5.5%) small deletion and 1 (5.5%) small duplication (Fig. 2). According to the standards developed by the American College of Medical Genetics and Genomics (ACMG) for the classification of these variants, seven variants were classified as pathogenic (P), six as a variant of uncertain significance (VUS), and five as likely pathogenic (LP). Novel variants comprised one P, seven LP, and three VUS.
These powerful gene-based strategies of diagnosis with WES provide a reliable tool, which helps clinicians to reduce the diagnostic problem commonly observed that exists due to the phenotypic variability and heterogeneity in most genetic NCL disease types.
Infantile and childhood forms of NCL are the most common forms of this disease, CLN3 disease (juvenile NCL) followed by CLN2 disease (classic late infancy), respectively, constitute the most common form of this disease in Western countries. CLN2 also is the most frequent in Southern Europe and the Mediterranean region. The uncommon forms of childhood NCL are CLN10, CLN12, and CLN14.
But in this study, the most mutations were in the CLN6 (41.3%) gene related to the CLN6 disease and after in the TPP1 (24.1%) gene related to the CLN2 disease. This nonconcurrence with previous estimates worldwide may be attributed to different factors: (a) differences in the genetic pool of Iranians due to racial diversity, (b) the absence of sufficient information on other sub-types of this disease or missing the patients due to faults of differential diagnosis and (c) small sample size is a potential limitation which may have introduced bias. Furthermore, some incompatibility in symptoms and phenotypic of our cases can be justified with this point that the classical form of each type of NCLs are generally correlated with the most common mutations in any gene, while unusual phenotypes and symptoms may arise from specific mutations and milder and later phenotypes may be due to so-called milder mutations or can be associated with related to the race of the patient and genetic differences caused by it. For example, mutation c.662C>T in the CLN6 gene was reported to associate with a more protracted progression of disease  (Sharp et al. 2003) but we found a family (F3) with three affected children with this mutation, and the progress of the disease in these patients was similar to other patients carrying other mutations in the CLN6 gene, we can explain this discrepancy with differences in the genetic pool. Further detailed research with more patients and longterm follow-up of patients will lead to progress in our awareness of these diseases, which is necessary to further understand the pathogenic mechanisms, a better understanding of genotype-phenotype correlation to improve the efficacy of genetic counseling, planning reasonable medical services and promote create new treatments.
Despite the limitations, we successfully identified 17 disease-causing variants, including 10 novel mutations using WES in 21 independent families. Our findings expand the spectrum of known mutations and their related clinical phenotypes in Iranian NCL patients.

Conclusion
We report the largest cohort of NCL disease patients in Iran so far. We identified 11 novel mutations and 7 other previously reported mutations (other ethnicities) in 23 Iranian families. It is hoped that the findings of this study will raise awareness for NCL disease and reduce the time from the start of first symptoms to diagnosis, helping families to choose prognostic counseling, prenatal diagnosis, and the best clinical therapeutic approach. Moreover, these data may further elucidate mechanisms underlying NCLs to help create more effective treatments.
Funding This study had no funding.
Data Availability All data found in this study from WES and Sanger sequencing, all primers we used, and all patients' information are available from the authors and are available from the corresponding author upon reasonable request.

Declarations
Conflict of interest Mrs. Samareh Panjeshahi declares that she has no conflict of interest. Professor Parvaneh Karimzadeh declares that she has no conflict of interest. Professor Abolfazl Movafagh declares that he has no conflict of interest. Professor Farzad Ahmadabadi declares that he has no conflict of interest. Dr. Elham Rahimian declares that she has no conflict of interest. Miss. Sahar Alijanpour declares that she has no conflict of interest. Professor Mohammad Miryounesi declares that he has no conflict of interest.
Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee of the Institutional Review Board (IRB), with the approval code of IR.SBMU.MSP.REC.1400. 250 and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent Informed consent was obtained from all individual participants included in the study.