A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

A needle in a haystack? The impact of a targeted epilepsy gene panel in the identification of a treatable but rapidly progressive metabolic epilepsy: CLN2 disease.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.

ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.

Clinical and Molecular Characteristics of Neuronal Ceroid Lipofuscinosis in Saudi Arabia.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCLs) represent a heterogeneous group of inherited metabolic lysosomal disorders characterized by neurodegeneration. This study sought to describe the clinical and molecular characteristics of NCLs in Saudi Arabia and determine the most common types in that population. METHODS: A retrospective review of electronic medical records was conducted for 63 patients with NCL (55 families) from six tertiary and referral centers in Saudi Arabia between 2008 and 2022. Clinical, radiological, and neurophysiological data as well as genetic diagnoses were reviewed. RESULTS: CLN6 was the predominant type, accounting for 45% of cases in 25 families. The most common initial symptoms were speech delay (53%), cognitive decline (50%) and/or gait abnormalities (48%), and seizure (40%). Behavioral symptomatology was observed in 20%, whereas visual impairment was less frequently (9.3%) encountered. Diffuse cerebral and cerebellar atrophy was the predominant finding on brain magnetic resonance imaging. Electroencephalography generally revealed background slowing in all patients with generalized epileptiform discharges in 60%. The most common genotype detected was the p.Ser265del variant found in 36% (20 of 55 families). The most rapidly progressive subtypes were CLN2 and CLN6. Two patients with each died at age five years. The earliest age at which a patient was nonambulatory was two years in a patient with CLN14. CONCLUSIONS: This is the largest molecularly confirmed NCL cohort study from Saudi Arabia. Characterizing the natural history of specific NLC types can increase understanding of the underlying pathophysiology and distinctive genotype-phenotype characteristics, facilitating early diagnosis and treatment initiation as well as genetic counseling for families.

Peripheral retinal finding on fluorescein angiography in neuronal ceroid lipofuscinosis type 2 (CLN2).

Classically, peripheral vascular changes in the retina in patients with neuronal ceroid lipofuscinosis type 2 (CLN2) are described as vascular attenuation seen in the late stages of disease on the Weill Connell Ophthalmic Severity Score (WCOSS) staging system. We describe isolated, mild, peripheral vasculitis with peripheral arteriolar dropout identified by fluorescein angiography in patients with a WCOSS grade of stage 2. We believe this vasculitis represents an early vasodegenerative phase of disease that leads to the vascular attenuation seen in later stages of the disease.

A computational approach to analyzing the functional and structural impacts of Tripeptidyl-Peptidase 1 missense mutations in neuronal ceroid lipofuscinosis.

Neuronal ceroid-lipofuscinosis (NCLs) are a group of severe neurodegenerative conditions, most likely present in infantile, late infantile, juvenile, and adult-onset forms. Their phenotypic characteristics comprise eyesight damage, reduced motor activity and cognitive function, and sometimes tend to die in the initial stage. In recent studies, NCLs have been categorized into at least 14 genetic collections (CLN1-14). CLN2 gene encodes Tripeptidyl peptidase 1 (TPP1), which affects late infantile-onset form. In this study, we retrieved a mutational dataset screening for TPP1 protein from various databases (ClinVar, UniProt, HGMD). Fifty-six missense mutants were enumerated with computational methods to perceive the significant mutants (G475R and G501C) and correlated with clinical and literature data. A structure-based screening method was initiated to understand protein-ligand interaction and dynamic simulation. The docking procedure was performed for the native (3EDY) and mutant (G473R and G501C) structures with Gemfibrozil (gem), which lowers the lipid level, decreases the triglycerides amount in the blood circulation, and controls hyperlipidemia. The Native had an interaction score of -5.57 kcal/mol, and the mutants had respective average binding scores of -6.24 (G473R) and - 5.17 (G501C) kcal/mol. Finally, molecular dynamics simulation showed that G473R and G501C mutants had better flexible and stable orientation in all trajectory analyses. Therefore, this work gives an extended understanding of both functional and structural levels of influence for the mutant form that leads to NCL disorder.

Intravitreal enzyme replacement for inherited retinal diseases.

PURPOSE OF REVIEW: This paper provides an update on intravitreal (IVT) enzyme replacement therapy (ERT) in metabolic retinal diseases; particularly neuronal ceroid lipofuscinosis type 2 (CLN2) also known as Batten disease. RECENT FINDINGS: ERT is being explored in CLN2 related Batten disease, a fatal neurodegenerative condition associated with retinopathy and blindness that is caused by the deficiency of lysosomal enzyme TPP1. Cerliponase alfa, a recombinant human tripeptidyl-peptidase1 (rhTPP1) administered by intraventricular infusions has been demonstrated to slow the rate of neurodegenerative decline but not retinopathy. A preclinical study of IVT rhTPP1 in a CLN2 canine model demonstrated efficacy in preserving retinal function and retinal morphology shown on histology. More recently, intravitreal (IVT) administration of rhTPP1 was reported in a first-in-human compassionate use study. Patients received 12-18 months of 8-weekly IVT ERT (0.2 mg rhTPP-1 in 0.05 ml) in one eye. No significant ocular adverse reactions were reported. Treatment decreased the rate of retinal thinning but modestly. SUMMARY: The evidence suggests that IVT ERT with rhTPP1 may be a safe and effective treatment for CLN2 retinopathy. However, the optimal dosage and frequency to achieve the best possible outcomes requires further investigation as does patient selection.

First in man study of intravitreal tripeptidyl peptidase 1 for CLN2 retinopathy.

BACKGROUND/OBJECTIVES: CLN2 Batten Disease is a fatal neurodegenerative condition of childhood associated with retinal dystrophy and blindness. Intracerebroventricular infusion of rhTPP1 greatly slows the rate of neurodegenerative decline but not retinopathy. Intravitreal rhTPP1 is known to slow retinal degeneration in a canine model of CLN2. We report a first-in-man controlled clinical trial of intravitreal rhTPP1 for CLN2 associated retinal dystrophy. SUBJECTS/METHODS: 8 children aged 5-9 with CLN2 Batten Disease were prospectively enroled. Severely affected patients were preferentially selected, provided that vision was better than no perception of light. Children underwent 8 weekly intravitreal injections of rhTPP1 (0.2 mg in 0.05 ml) into the right eye for 12-18 months. The left eye was untreated and acts as a paired control. The primary outcome was safety based on the clinical detection of complications. A secondary outcome was paracentral macular volume (PMV) measured by spectral domain OCT. Linear regression/paired t tests were used to compare rates of decline. RESULTS: No severe adverse reactions (uveitis, raised IOP, media opacity) occurred. The mean baseline PMV was 1.28 mm3(right), 1.27 mm3(left). 3 of the youngest patients exhibited bilateral progressive retinal thinning (p < 0.05), whereas retinal volume was stable in the remaining 5 patients. In the 3 patients undergoing retinal degeneration, the rate of PMV loss was slower in the treated vs. untreated eye (p = 0.000042, p = 0.0011, p = 0.00022). CONCLUSIONS: Intravitreal rhTPP1 appears to be a safe and effective treatment for CLN2 related retinopathy however commencement of treatment early in the course of disease is more likely to be efficacious.

Safety and efficacy of cerliponase alfa in children with neuronal ceroid lipofuscinosis type 2 (CLN2 disease): an open-label extension study.

BACKGROUND: Cerliponase alfa is a recombinant human tripeptidyl peptidase 1 (TPP1) enzyme replacement therapy for the treatment of neuronal ceroid lipofuscinosis type 2 (CLN2 disease), which is caused by mutations in the TPP1 gene. We aimed to determine the long-term safety and efficacy of intracerebroventricular cerliponase alfa in children with CLN2 disease. METHODS: This analysis includes cumulative data from a primary 48-week, single-arm, open-label, multicentre, dose-escalation study (NCT01907087) and the 240-week open-label extension with 6-month safety follow-up, conducted at five hospitals in Germany, Italy, the UK, and the USA. Children aged 3-16 years with CLN2 disease confirmed by genetic analysis and enzyme testing were eligible for inclusion. Treatment was intracerebroventricular infusion of 300 mg cerliponase alfa every 2 weeks. Historical controls with untreated CLN2 disease in the DEM-CHILD database were used as a comparator group. The primary efficacy outcome was time to an unreversed 2-point decline or score of 0 in the combined motor and language domains of the CLN2 Clinical Rating Scale. This extension study is registered with ClinicalTrials.gov, NCT02485899, and is complete. FINDINGS: Between Sept 13, 2013, and Dec 22, 2014, 24 participants were enrolled in the primary study (15 female and 9 male). Of those, 23 participants were enrolled in the extension study, conducted between Feb 2, 2015, and Dec 10, 2020, and received 300 mg cerliponase alfa for a mean of 272·1 (range 162·1-300·1) weeks. 17 participants completed the extension and six discontinued prematurely. Treated patients were significantly less likely than historical untreated controls to have an unreversed 2-point decline or score of 0 in the combined motor and language domains (hazard ratio 0·14, 95% CI 0·06 to 0·33; p<0·0001). All participants experienced at least one adverse event and 21 (88%) experienced a serious adverse event; nine participants experienced intracerebroventricular device-related infections, with nine events in six participants resulting in device replacement. There were no study discontinuations because of an adverse event and no deaths. INTERPRETATION: Cerliponase alfa over a mean treatment period of more than 5 years was seen to confer a clinically meaningful slowing of decline of motor and language function in children with CLN2 disease. Although our study does not have a contemporaneous control group, the results provide crucial insights into the effects of long-term treatment. FUNDING: BioMarin Pharmaceutical.

CLN2 disease resulting from a novel homozygous deep intronic splice variant in TPP1 discovered using long-read sequencing.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative disorder with enzyme replacement therapy available. We present two siblings with a clinical diagnosis of CLN2 disease, but no identifiable TPP1 variants after standard clinical testing. Long-read sequencing identified a homozygous deep intronic variant predicted to affect splicing, confirmed by clinical DNA and RNA sequencing. This case demonstrates how traditional laboratory assays can complement emerging molecular technologies to provide a precise molecular diagnosis.

Acidified drinking water improves motor function, prevents tremors and changes disease trajectory in Cln2R207X mice, a model of late infantile Batten disease.

Batten disease is a group of mostly pediatric neurodegenerative lysosomal storage disorders caused by mutations in the CLN1-14 genes. We have recently shown that acidified drinking water attenuated neuropathological changes and improved motor function in the Cln1R151X and Cln3-/- mouse models of infantile CLN1 and juvenile CLN3 diseases. Here we tested if acidified drinking water has beneficial effects in Cln2R207X mice, a nonsense mutant model of late infantile CLN2 disease. Cln2R207X mice have motor deficits, muscle weakness, develop tremors, and die prematurely between 4 and 6 months of age. Acidified water administered to Cln2R207X male mice from postnatal day 21 significantly improved motor function, restored muscle strength and prevented tremors as measured at 3 months of age. Acidified drinking water also changed disease trajectory, slightly delaying the death of Cln2R207X males and females. The gut microbiota compositions of Cln2R207X and wild-type male mice were markedly different and acidified drinking water significantly altered the gut microbiota of Cln2R207X mice. This suggests that gut bacteria might contribute to the beneficial effects of acidified drinking water. Our study demonstrates that drinking water is a major environmental factor that can alter disease phenotypes and disease progression in rodent disease models.

Language Delay in Patients with CLN2 Disease: Could It Support Earlier Diagnosis?

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is a rare pediatric disorder associated with rapid neurodegeneration, and premature death in adolescence. An effective enzyme replacement therapy (cerliponase alfa) has been approved that can reduce this predictable neurological decline. The nonspecific early symptoms of CLN2 disease frequently delay diagnosis and appropriate management. Seizures are generally recognized as the first presenting symptom of CLN2 disease, but emerging data show that language delay may precede this. An improved understanding of language deficits in the earliest stage of CLN2 disease may support the early identification of patients. In this article, CLN2 disease experts examine how language development is affected by CLN2 disease in their clinical practices. The authors' experiences highlighted the timings of first words and first use of sentences, and language stagnation as key features of language deficits in CLN2 disease, and how deficits in language may be an earlier sign of the disease than seizures. Potential challenges in identifying early language deficits include assessing patients with other complex needs, and recognizing that a child's language abilities are not within normal parameters given the variability of language development in young children. CLN2 disease should be considered in children presenting with language delay and/or seizures to facilitate earlier diagnosis and access to treatment that can significantly reduce morbidity.

Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.

Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy-lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition.

Clinical management and diagnosis of CLN2 disease: consensus of the Brazilian experts group.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare neurodegenerative genetic disease that affects children in early life. Its classic form is rapidly progressive, leading to death within the first 10 years. The urge for earlier diagnosis increases with the availability of enzyme replacement therapy. A panel of nine Brazilian child neurologists combined their expertise in CLN2 with evidence from the medical literature to establish a consensus to manage this disease in Brazil. They voted 92 questions including diagnosis, clinical manifestations, and treatment of the disease, considering the access to healthcare in this country. Clinicians should suspect CLN2 disease in any child, from 2 to 4 years old, with language delay and epilepsy. Even though the classic form is the most prevalent, atypical cases with different phenotypes can be found. Electroencephalogram, magnetic resonance imaging, molecular and biochemical testing are the main tools to investigate and confirm the diagnosis. However, we have limited access to molecular testing in Brazil, and rely on the support from the pharmaceutical industry. The management of CLN2 should involve a multidisciplinary team and focus on the quality of life of patients and on family support. Enzyme replacement therapy with Cerliponase α is an innovative treatment approved in Brazil since 2018; it delays functional decline and provides quality of life. Given the difficulties for the diagnosis and treatment of rare diseases in our public health system, the early diagnosis of CLN2 needs improvement as enzyme replacement therapy is available and modifies the prognosis of patients.

Lipofuscinose ceróide neuronal (CLN2) é uma doença genética neurodegenerativa rara que afeta crianças nos primeiros anos de vida. A sua forma clássica é rapidamente progressiva, levando à morte nos primeiros 10 anos. A necessidade de um diagnóstico precoce aumenta com a disponibilidade do tratamento de terapia enzimática. Um painel de nove neurologistas infantis brasileiros combinou sua experiência em CLN2 com evidências da literatura médica para estabelecer um consenso no manejo desta doença no Brasil. Eles votaram 92 questões abordando diagnóstico, manifestações clínicas e tratamento, considerando o acesso à saúde no Brasil. Deve-se suspeitar de CLN2 em qualquer criança de 2 a 4 anos de idade que apresente atraso de linguagem e epilepsia. Apesar da forma clássica ser a mais prevalente, podem ser encontrados casos atípicos com diferentes fenótipos. Eletroencefalograma, ressonância magnética, testes moleculares e bioquímicos são as principais ferramentas para investigar e confirmar o diagnóstico. No entanto, o acesso aos testes moleculares é limitado no Brasil, necessitando contar com o apoio da indústria farmacêutica. O manejo da CLN2 deve envolver uma equipe multidisciplinar e focar na qualidade de vida dos pacientes e no apoio familiar. A terapia de reposição enzimática com Cerliponase alfa é um tratamento inovador aprovado no Brasil desde 2018; ele retarda o declínio funcional e proporciona qualidade de vida. Diante das dificuldades para o diagnóstico e tratamento de doenças raras em nosso sistema público de saúde, o diagnóstico precoce de CLN2 precisa de melhorias pois a terapia de reposição enzimática está disponível e modifica o prognóstico dos pacientes.

A mouse mutant deficient in both neuronal ceroid lipofuscinosis-associated proteins CLN3 and TPP1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3, respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2, and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1.

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.

Magnetic resonance brain volumetry biomarkers of CLN2 Batten disease identified with miniswine model.

Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease (Batten disease) is a rare pediatric disease, with symptom development leading to clinical diagnosis. Early diagnosis and effective tracking of disease progression are required for treatment. We hypothesize that brain volumetry is valuable in identifying CLN2 disease at an early stage and tracking disease progression in a genetically modified miniswine model. CLN2R208X/R208X miniswine and wild type controls were evaluated at 12- and 17-months of age, correlating to early and late stages of disease progression. Magnetic resonance imaging (MRI) T1- and T2-weighted data were acquired. Total intercranial, gray matter, cerebrospinal fluid, white matter, caudate, putamen, and ventricle volumes were calculated and expressed as proportions of the intracranial volume. The brain regions were compared between timepoints and cohorts using Gardner-Altman plots, mean differences, and confidence intervals. At an early stage of disease, the total intracranial volume (- 9.06 cm3), gray matter (- 4.37% 95 CI - 7.41; - 1.83), caudate (- 0.16%, 95 CI - 0.24; - 0.08) and putamen (- 0.11% 95 CI - 0.23; - 0.02) were all notably smaller in CLN2R208X/R208X miniswines versus WT, while cerebrospinal fluid was larger (+ 3.42%, 95 CI 2.54; 6.18). As the disease progressed to a later stage, the difference between the gray matter (- 8.27%, 95 CI - 10.1; - 5.56) and cerebrospinal fluid (+ 6.88%, 95 CI 4.31; 8.51) continued to become more pronounced, while others remained stable. MRI brain volumetry in this miniswine model of CLN2 disease is sensitive to early disease detection and longitudinal change monitoring, providing a valuable tool for pre-clinical treatment development and evaluation.

Economic analysis of cerliponase alfa for treatment of late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2).

BACKGROUND: Cerliponase alfa is an orphan drug approved for the treatment of late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2). AIM: Our goal was to assess the cost-effectiveness of cerliponase alfa in patients with CLN2 in the socioeconomic context of the Republic of Serbia in contrast to symptomatic therapy. METHOD: For this study, a forty-year horizon and the perspective of the Serbian Republic Health Insurance Fund were used. Quality-adjusted life years gained with cerliponase alfa and comparator, as well as direct treatment costs, were the study's key outcomes. The creation and simulation of a discrete-event simulation model served as the basis for the investigation. Monte Carlo microsimulation was performed on a sample of 1000 virtual patients. RESULTS: When compared to symptomatic therapy, cerliponase alfa treatment was not cost-effective and was linked to negative net monetary benefit regardless of when the illness signs started. CONCLUSION: Cerliponase alfa is not more economical than symptomatic therapy for the treatment of CLN2 when using typical pharmacoeconomic analysis. Cerliponase alfa has been shown to be effective but more has to be done to make it accessible to all CLN2 patients.

Cynomolgus macaque model of neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degeneration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a homozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

Intravitreal gene therapy preserves retinal function in a canine model of CLN2 neuronal ceroid lipofuscinosis.

CLN2 neuronal ceroid lipofuscinosis is a rare hereditary neurodegenerative disorder characterized by deleterious sequence variants in TPP1 that result in reduced or abolished function of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). Children with this disorder experience progressive neurological decline and vision loss starting around 2-4 years of age. Ocular disease is characterized by progressive retinal degeneration and impaired retinal function culminating in total loss of vision. Similar retinal pathology occurs in a canine model of CLN2 disease with a null variant in TPP1. A study using the dog model was performed to evaluate the efficacy of ocular gene therapy to provide a continuous, long-term source of human TPP1 (hTPP1) to the retina, inhibit retinal degeneration and preserve retinal function. TPP1-/- dogs received an intravitreal injection of 1 x 1012 viral genomes of AAV2.CAG.hTPP1 in one eye and AAV2.CAG.GFP in the contralateral eye at 4 months of age. Ophthalmic exams, in vivo ocular imaging and electroretinography were repeated monthly to assess retinal structure and function. Retinal morphology, hTPP1 and GFP expression in the retina, optic nerve and lateral geniculate nucleus, and hTPP1 concentrations in the vitreous were evaluated after the dogs were euthanized at end stage neurological disease at approximately 10 months of age. Intravitreal administration of AAV2.CAG.hTPP1 resulted in stable, widespread expression of hTPP1 throughout the inner retina, prevented disease-related declines in retinal function and inhibited disease-related cell loss and storage body accumulation in the retina for at least 6 months. Uveitis occurred in eyes treated with the hTPP1 vector, but this did not prevent therapeutic efficacy. The severity of the uveitis was ameliorated with anti-inflammatory treatments. These results indicate that a single intravitreal injection of AAV2.CAG.hTPP1 is an effective treatment to inhibit ocular disease progression in canine CLN2 disease.