Identification of a TPP1 Q278X Mutation in an Iranian Patient with Neuronal Ceroid Lipofuscinosis 2: Literature Review and Mutations Update

Neuronal ceroid lipofuscinoses type 2 (CLN2), the most common form of Batten disease, is caused by TPP1 loss of function, resulting in tripeptidyl peptidase-1 enzyme deficiency and cerebral accumulation of lipopigments. Clinical hallmarks include epileptic seizures, vision loss, progressive movement disorder, ataxia, and eventually death. Diagnosis is often delayed due to the rarity of the conditions. Results: Here, we report a case presenting with clinical features of CLN2, carrying a homozygous novel nonsense variant in TPP1 (NM_000391:c.C832T, (p.Q278*), rs1352347549). Moreover, we performed a comprehensive literature review regarding previously identified disease-causing TPP1 mutations and genotype-phenotype correlations. Conclusion: Depending on the type of mutation, different phenotypes are observed in patients with CLN2, suggesting that the severity of phenotypes is related to the genotype of the patients.


Introduction
Neuronal ceroid lipofuscinoses (NCLs) represent a group of rare clinically and genetically heterogeneous progressive neurodegenerative lysosomal storage disorders (LSD), mainly affecting children aged 2-6 years, caused by loss-of-function mutations in CLNgenes. With the exception of CLN4 which is autosomal-dominantly inherited, all other forms follow an autosomal-recessive mode of inheritance [1][2][3]. Children mostly show normal psychomotoric development until progressive development of seizures, vision loss, intellectual and motor decline, cognitive impairment, and eventually premature death. Despite the rarity of this disease, 300-350 cases of NCLs are reported annually in the US, and its global incidence is one per 100,000 live births (orphaned; ORPHA 79264). The highest incidence rates have been reported in some Nordic countries (one per 14,000 in Iceland) [1,4,5], while incidence is unknown for many other countries, including Iran.
CLN2 (OMIM#607998) is located on the short arm of chromosome 11 (11p15), consisting of 13 exons and 12 introns [5,[15][16][17][18]. CLN2 encodes for Tripeptidyl Peptidase 1 (TPP1), a lysosomal aminopeptidase cleaving N-terminal tripeptides from small polypeptides. In humans, this gene encodes a 61-kDa inactive precursor (proenzyme) containing 563 amino acids (aa), including a 368-aa catalytic domain, a 19-aa leader sequence (signal peptide), and a 176-aa prodomain. A signal peptide enables ER-specific localization for cotranslational cleavage in the ER lumen. This is followed by N-glycosylation at the Golgi, enabling proper folding, enhancing protein stability, activity, and intracellular enzyme targeting. Transport to the lysosome is enabled by mannose-6-phosphate receptor binding. At the lysosome, CLN2 is first further processed into a 50 kDa polypeptide followed by cleavage into the mature 368 amino acid (48 kDa) enzyme by lysosomal exoglycosidases such as neuraminidases [19][20][21]. Lysosomal degradation pathways involving TPP1 are summarized in Figure 2. Overlapping clinical features and extensive genetic heterogeneity renders next-generation sequencing (NGS) the most efficient and precise genetic diagnostic method if NCL is suspected. In the present study, we describe the identification of a novel homozygous TPP1 variant in an Iranian patient with CLN2 and present an overview of previously published CLN2 disease alleles as well as genotype-phenotype correlations.

Materials and Methods
The research ethics committee ethically approved this study of the Mashhad University of medical sciences committee, and all study participants signed the written informed consent (IR.MUMS.REC.1395.534). Genetic diagnostics was performed under the Radboudumc Innovative Genetics Diagnostics program.

DNA Extraction
Genomic DNA was extracted from the peripheral blood of the proband and his family members using the established salting-out method, and the integrity of the extracted DNA was assessed by gel electrophoresis (1% agarose).

Whole Exome Sequencing
Two microgrammes of DNA of the proband was subjected to exome capture using Agilent SureSelect Human All Exon V6 Kit (Agilent, Sata Clara, CA, USA) and sequencing performed on an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) sequencer for an average 50× sequencing depth, resulting in sequences of greater than 100 bases from each end of the fragments (Novogene, Cambridge, UK). GATK-based pipeline [22] was used for variant calling using Burrows-Wheeler alignment [23] to perform sequence alignment to the GRCh37/UCSC hg19 reference genome. SNV were detected using VarScan version 2.2.5, MuTec, Iranome, gnomAD, and Greater Middle East (GME) Variome Project databases were used for population-frequency specific variant filtering.

Polymerase Chain Reaction (PCR) and Sanger sequencing
We used 1 µL of genomic DNA for PCR reactions. Furthermore, 50 pmol of each forward (5 -GGGATCACTGTGGAGTCAAAG-3 ) and reverse (5 -AGACCTGGCTCAGTTCATGC-3 ) primers, 10 µL of 2× PCR Master Mix (Sina Clon Cat. No.: MM2062), and 12 µL of distilled water were used in a final volume of 25 µL. PCR reactions were carried out using an initial incubation step at 95 • C for 5 min followed by 35 cycles at 94 • C for 40 s, annealing at 60 • C for 40 s, and extension at 72 • C for 40 s. A final extension step at 72 • C for 10 min was used. We analyzed 5 µL of PCR products using a 2% agarose gel for electrophoresis. Subsequently, the PCR products were purified and sequenced using Sanger sequencing.

Literature Search
Search terms "TPP1" or "CLN2" were used in PubMed to retrieve relevant human mutation reports and ClinVar and ACMG databases were searched for reported TPP1 alleles.

Clinical Assessment
A 5-year-old patient diagnosed with epilepsy and ataxia was referred to our clinic after a generalized tonic-clonic seizure. The boy was born to a consanguineous Iranian family (parents were first cousins) with an unremarkable family history for neurological disorders; however, the mother experienced two miscarriages due to unknown reasons at 12 and 14 weeks of gestation ( Figure 3A). The patient was born naturally at term with a birth weight of 3.2 kg. Perinatal course and development were normal. Motor and developmental milestones were reached at normal age, for example, free walking at 12 months and talking in sentences at 24 months. At four years of age, a generalized tonic-clonic seizure occurred followed by rapid cognitive and motoric regression, including losing the ability to walk, loss of speech, progressive ataxia, spasticity, and difficulties swallowing. In the last examination, the patient was found to be legally blind. Hematology tests revealed mild anemia (erythroctyes 4.48/µL, hematocrit 32.6%, and hemoglobin 10.6 g/dL). Serum glutamic-oxaloacetic transaminase and alkaline phosphatase were increased (46 mg/dL and 430 U/L, respectively). Mag-netic resonance imaging (MRI) findings revealed only mild supratentorial dilation of the ventricular system. There was cerebellar vermian and significant cerebellar hemispheres hypoplasia with prominent 4th ventricle and cistern magna, thinning of superior cerebellar peduncles and cystic lesion of cerebrospinal fluid (CSF) intensity communication with 4th ventricle, and delayed myelinisation of the periventricular white matter and centrum semiovale ( Figure 4). The patient underwent chest physiotherapy and received Levetiracetam, Sodium valproate, Nitrazepam, Tetracosactide, Vitamin B6, and Biotin. At the age of 4 years and 8 months, the patient was admitted to the ICU with a GCS of 4.5 and died after 2 months.

Genetic Analysis
Whole-exome sequencing and analysis revealed a novel homozygous loss of function (LOF) variant in exon 7 of TPP1 (c.C832T: p.Gln278Ter, rs135234754). The allelic frequency of this variant in the gnomAD database is 0.000003977 while no frequency has been reported in ExAC or the 1000 genomes databases. The homozygous variant co-segregated with the disease in family, with both parents and a healthy sibling found to be heterozygous carriers ( Figure 3B-E).

Discussion
CLN2 is a specific subtype of NCLs caused by TPP1 loss of function, resulting in seizures, vision loss, dementia, cerebellar ataxia, sleep disorders, progressive psychomotor decline, and death in the first decade of life [24]. Early symptoms of CLN2 include seizures and photosensitivity [25]; however, seizures can rarely be absent [3,26]. In line with these findings, our case initially presented with a generalized tonic-clonic seizure. Microcephaly, absent in our case, has additionally been reported in a few cases, including a 9-yearold Australian boy [27] and an 8-year-old boy from Turkey [7], both of whom had nonconsanguineous parents. Further common findings include cerebellar atrophy, resulting in ataxia and/or tremor including in our case [7,13,[28][29][30][31] (Table 1). Further, vision loss and/or optic atrophy was observed in most patients. Further frequent manifestations include spasticity as well as swallowing and sleeping problems. Based on PubMed publications and submissions in ClinVar, more than 155 potentially disease-associated TPP1 variants have been reported in total (Supplementary Table S1 and Figure 5). TPP1 is required in lysosomes for protein degradation and loss of TPP1 activity results in the accumulation of auto fluorescent lysosomal storage material in various cell types, including neurons [39]. Overall, missense variants are most frequently identified (63, 48%), followed by frameshift (21, 16%), and stop-gain (17, 13%) variants [6,17]. In addition to CLN2, TPP1 dysfunction can also result in autosomal-recessive Spinocerebellar Ataxia 7 (MIM609270) [2,32]. This phenotype is characterized by cerebellar ataxia, tremor, dysarthria, and nystagmus with an age of onset during the first or second life decade. Likely, alleles causing spinocerebellar ataxia result on proteins with some residual function, hence the hypomorphic phenotype in comparison to CLN2 disease alleles with later age of onset and an overall less severe clinical picture [40]. In line with this, missense alleles can be frequently identified in Spinocerebellar Ataxia 7, while approximately 60% of patients with CLN2 carry two common LOF mutations: the splice acceptor site mutation (c.509-1G > C) and the stop-gain mutation in exon 6 (c.622C > T, p.R208X), which may occur homozygously or heterozygously in trans (compound-heterozygously) with other disease alleles [1,8,41]. In the present study, we report a novel homozygous pathogenic stop-gain variant (p.Arg278*) in an Iranian family. This allele results in a truncation within the peptidase S_53 domain. Up to now, 73 TPP1 null variants have been reported in the ClinVar, six of which are found in exon 7. The role of genetic diagnostics in CLN, especially CLN2, has become more important than ever. In pediatric cases of CLN2, replacement of the dysfunctional TPP1 enzyme with a functional recombinant enzyme (Cerliponase alpha) by intraventricular injection has been reported to effectively delay disease progression and to stabilize the loss of motor and language function [24,41,42]. To achieve the best possible outcomes, an early genetic diagnosis will be essential. For our case, diagnosis came too late for possible interventions.
Further, genetic testing enables prenatal and pre-implantation diagnostics as well as carrier identification, offering novel family planning options.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11216415/s1, Table S1: TPP1 variants identified in CLN2 patients. Informed Consent Statement: Informed consent was obtained from all subjects or their legal guardians involved in the study.

Data Availability Statement:
Sequencing data is available on personal request for reasonable purposes.