|Year : 2018 | Volume
| Issue : 1 | Page : 11-18
Assessment of phenotype and genotype in neuronal ceriod lipofuscinosis among Egyptian children
Hisham Megahed PhD.,MD 1, Ekram Fateen1, Hamed El-Gawaby2, Nadia Galal3, Amina Hindawy2
1 Department of Clinical Genetics, The National Research Centre, Division of Medical Genetics and Genome Research, Giza, Egypt
2 Department of Paediatrics; Department of Radiology, Cairo University, Giza, Egypt
3 Department of Neurology, Division of Electron Microscope, Ain Shams University, Cairo, Egypt
|Date of Submission||31-Oct-2017|
|Date of Acceptance||17-Dec-2017|
|Date of Web Publication||26-Mar-2018|
2 Aziz Osman Street, Flat 156, Zamalek, Cairo
Source of Support: None, Conflict of Interest: None
Neuronal ceroid lipofuscinosis (NCLs) is a group of recessively inherited neurodegenerative disorder. It is a lysosomal storage disease caused by intracellular accumulation of autoflorescent lipofuscin pigments. The aim of this study is to compare the clinical, molecular genetics, neurophysiological, neuroradiological, and ultrastructural characteristics of NCLs among Egyptian patients with patients with the same disorder in other populations.
Patients and methods
Our cohort included 32 Egyptian NCL patients. There were 20 males and 12 females. Their ages ranged from 1.9 to 4.6 years, with a mean age of 3.5 years. Our clinically diagnosed cohort of NCLs was subjected to neurological, neurophysiological, neuroradiological, and ultrastructural studies as well as whole-exome sequencing (WES) to confirm the diagnosis.
Our cases were compared with the clinical, neurophysiological, neuroradiological, and ultrastructural characteristics as well as molecular genetics of NCL patients worldwide. Our cases presented clinically with developmental delay, ataxia followed by seizures, and eventually a vegetative state. Their MRIs of the brain showed mainly cerebellar atrophy with or without cortical brain atrophy. Our results showed that the late infantile type of NCL is the most common clinical type among our Egyptian cases. WES showed that CLN6 is the most common mutated gene in our cohort.
Our results were analyzed and compared with the characteristics of other patients with NCL worldwide.
Expanding the studies and research on NCL patients will further identify the diversity and the molecular genetics involved in the pathogenesis of this disorder. Subsequently, this will lead to potential identification of therapeutic and prenatal diagnostic methods and preventive measures.
Keywords: brain atrophy, developmental delay, gingival biopsy, neuronal ceroid lipofuscinosis, whole exome sequencing
|How to cite this article:|
Megahed H, Fateen E, El-Gawaby H, Galal N, Hindawy A. Assessment of phenotype and genotype in neuronal ceriod lipofuscinosis among Egyptian children. Middle East J Med Genet 2018;7:11-8
|How to cite this URL:|
Megahed H, Fateen E, El-Gawaby H, Galal N, Hindawy A. Assessment of phenotype and genotype in neuronal ceriod lipofuscinosis among Egyptian children. Middle East J Med Genet [serial online] 2018 [cited 2021 Jan 22];7:11-8. Available from: http://www.mxe.eg.net/text.asp?2018/7/1/11/228077
| Introduction|| |
Neuronal ceroid lipofuscinosis (NCL) includes a group of progressive neurodegenerative disorders affecting mainly infants and children. The frequency of NCL varies according to race and geographic distributions. It is estimated to be 1 : 12 500 live births in Anglo-Saxon countries (Chabrol et al., 2013), whereas it has an incidence of 1.6–2.4/100 000 in USA (Mole et al., 2011).
NCLs are genetically inherited autosomal recessive disorders caused by one of several heterogeneous genes.
They are lysosomal storage diseases caused by intracellular accumulation of autoflorescent lipofuscin pigment (Mink et al., 2013). Cells accumulate storage material known as granular osmophilic deposits (GRODs).
To date, at least 14 affected genes have been found to be involved. These include CLN1–CLN14. Thirteen of these are known (CLN1–CLN8 and CLN10–CLN14); however, CLN9 refers to the predicted locus in a family that does not appear to have mutations in any of the known genetic foci (Schulz et al., 2004).
NCLs are classified according to the age of clinical onset of symptoms as infantile, late infantile, and juvenile. Molecular and biochemical researches have broadened this classification on the basis of identification of several genetics foci. Infantile NCL is caused by genetic mutations in palmitoyl protein thioestrase (PPT-1), a lysosomal serine lipase. Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by genetic mutations in tripeptidyl peptidase, which also codes for a soluble lysosomal enzyme whose substrates are unknown (Schulz et al., 2013).
Clinically, NCLs cause a progressive delay in motor and mental milestones, leading to unsteadiness of gait, ataxia, epilepsy, and dementia. Retinal degeneration may lead to blindness. Nearly all forms of NCLs result in death despite trials by researchers exploring several therapeutic strategies.
Although NCLs are widely encountered worldwide, there are very few studies in Egyptian patients.
We believe that NCLs are frequent amongst Egyptian children as a result of the high rate of consanguineous marriages.
Therefore, the aim of this study is to investigate the association of the clinical, genetic, neuroradiological, and ultrastructural characteristics of NCLs among Egyptian patients and compare them with patients with the same disorder in other populations.
| Patients and Methods|| |
Thirty-two Egyptian children were included in this study.
There were 20 males and 12 females, with a male: female ratio of 5:3. Their ages ranged from 1.9 to 4.6 years, with a mean age of 3.5 years.
A written informed consent was obtained from all parents or care givers to carry out this study.
The diagnosis in the participants was made on the basis of clinical and neurological examinations. All cases were subjected to neurophysiological tracings including electroencephalography (EEG), visual evoked response, and retinogram.
Also, all cases underwent MRI of the brain.
In 18 cases, the diagnosis was confirmed by an ultrastructural electron microscopic histopathological study of their gingival biopsy to visualize the intracellular accumulation of the autoflorescent lipofuscin pigment.
However, the diagnosis of another 14 cases was confirmed by subjecting their blood samples to whole-exome sequencing (WES) to detect the genes responsible; eight cases showed mutation abnormalities in the CLN6 gene, four cases had an abnormality in the CLN7 gene, and only two cases showed an abnormality in the CLN2 gene.
Twenty-one cases were subjected to an enzymatic analysis to determine their diagnosis.
EEG analysis was carried out under standard conditions with hyperventilation and photic stimulation for all the cases as per protocol (Scaioli and Nardocci, 2000).
Visual evoked potential
Visual evoked potential (VEP) examination was performed for each eye separately using a flash frequency of 8 Hz to test for reproducibility of the responses.
Flash electroretinography (ERG) examination was performed on both eyes under photopic conditions (Vantianen et al., 1997).
High-coverage WES was used (>97% of the cases was sequenced at least 30 ×). This technique was used to genetically explore a cohort of 14 Egyptian patients. WES data were analyzed using filtering for dominant or recessive inheritance patterns.
Electron-microscopic examination of gingival biopsy
Gingival biopsy was examined by electron microscopy as per protocol (Mole and Williams, 2013).
Enzymatic assay was carried on peripheral blood lymphocytes to assess PPT and tripeptidyl peptidase enzymes as per protocol (Voznyi et al., 1999).
| Results|| |
[Table 1] shows the clinical findings of our 32 Egyptian children diagnosed with NCL. There were 20 males and 12 females, with a male:female ratio of 5:3. Their ages at onset of the disease ranged from 1.9 to 4.6 years, with a mean age of 3.5 years.
|Table 1: Clinical, neurophysiological, pathological, and genetic studies amongst our neuronal ceroid lipofuscinosis Egyptian patients|
Click here to view
All cases had positive consanguinity.
Our cases include 10 children with infantile NCL, whereas the rest of the 22 cases had LINCL, with an incidence of 68.75% of late type among our cases as shown in [Figure 1]. No juvenile cases were detected among our patients.
Cases with infantile NCL presented clinically with motor developmental delay, followed by seizures and later on global developmental delay and dementia.
Twelve cases of LINCL started clinically with titubation and ataxic gait, followed by akinetic fits, whereas the rest of the 10 cases of LINCL presented clinically with seizures, followed by ataxia, motor developmental deterioration, and finally dementia.
All our cases developed seizures at different stages of the disease. Seizures started with myoclonic epilepsy, followed by generalized tonic–clonic convulsions and akinetic fits, in some cases leading to a vegetative state.
EEG was carried under standard conditions with hyperventilation and photic stimulations. All cases showed background activities of 9–10 c/s α waves with fast waves that were nearly symmetrical and synchronous on both hemispheres and occipital regions.
Visual evoked potential
VEP showed evidence of abnormality in the visual pathway in 80% of the cases.
Flash ERG showed evidence of abnormalities of the rods and cons-mediated functions in the right eye in 85% of cases and in the left eye in 65% of cases.
Only 14 of our Egyptian patients were subjected to WES, which indicated that eight cases had mutations in the CLN6 gene, four cases showed mutations in the CLN7 gene, and only two cases showed CLN2 gene mutations as shown in [Table 1].
MRI of the brain was performed for all cases. [Table 2] shows MRI changes in our 32 Egyptian patients with NCLs.
|Table 2: MRI findings of the brain in our neuronal ceroid lipofuscinosis Egyptian patients|
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Twenty-eight cases showed cerebellar atrophy (87.5%). Eighteen out of these 28 cases had hemispherical as well as vermal cerebellar atrophy.
All cases with LINCLs showed cerebellar atrophy as shown in [Figure 2].
Twenty cases had overlapping cortical cerebral atrophy with or without ventriculomegaly.
Periventricular hypodensities on T2-weighted images were present in 16 (56%) cases, whereas basal ganglia involvement could be detected in six (18.75%) cases and only two cases showed thinning of the corpus callosum as shown in [Figure 3].
No MRI changes could be detected in the brain stem in our Egyptian cases.
Eighteen cases were subjected to ultrastructural examination of their gingival biopsies by an electron microscope to detect the presence of intracellular florescent lipofuscin pigment. All these cases showed membrane-bound vacuoles within the cytoplasm of fibroblasts. These vacuoles contain ill-defined curvy-linear structures known as GROD, indicating the presence of lipofuscin florescent pigments as shown in [Figure 4].
|Figure 4: Electron microscopic picture of the gingival biopsy of case No. 5|
Click here to view
Twenty-one blood samples from our cases was subjected to an enzymatic assay analysis and proved to have low enzymatic levels of the enzymes PPT and PTT.
| Discussion|| |
NCL are a heterogeneous group of autosomal recessive genetically inherited disorders. They are the most common cause of progressive neurodegenerative disorders in children (Nita et al., 2016).
The prevalence of the types of NCL depends on the race and country of origin, which is because of genetic variations (Jalanko and Braulke, 2009).
They are characterized by progressive degeneration of the gray matter of the brain, including the cortex, grey nuclei of the cerebellum, and retina (Mink et al., 2013).
As a result of the high rate of consanguineous marriages in Egypt, we believe that NCLs are frequent in Egyptian children. However, there is still no information on NCL in Egypt. Therefore, further clinical, biochemical, and molecular genetic studies are essential to understand the genetic heterogeneity of this disorder.
This study was carried out on 32 Egyptian children diagnosed clinically with NCLs.
Their diagnosis was confirmed by neurophysiological, neuroradiological, and ultrastructural studies. In some cases, WES was performed to detect the CLN gene mutations responsible.
All cases were products of consanguineous marriages. Males were more affected than females.
Late infantile type was the most prevalent in our series, found in 68.75% of the patients. This is similar to the study carried out by Topcu et al. (2004), who reported that LINCL and its Turkish variant were present in 77% of their patients. However, our results are in contrast with those of the European series in which it was reported that the juvenile type was the most frequent (Claussen et al., 1992).
In contrast, in Finland and Italy, the infantile type was the most frequent (Greaves J, Chamberlain LH 2007). Oishi et al. (1999) reported that LINCL and juvenile types of NCL were the most frequent types in Japan, which is in accordance with the results found in European countries. This indicates that the clinical distributions of types of NCL amongst our cases are similar to those in Turkey, but different from those in European countries and Japan. These results indicate that the variation in the clinical spectrum of NCL types in different parts of the world could be attributed to genetic diversity.
Seizures were the main clinical presentation among our 32 Egyptian children with NCLs. Our patients presented clinically with generalized tonic–clonic seizures, soon followed by myoclonic epilepsy and akinetic fits.
Fourteen out of 22 (63.6%) of our LINCL presented clinically with seizures, followed by ataxic manifestations, and later on they developed motor delay and a vegetative state. However, four out of our 22 cases of NINCL (18.2 %) started clinically by ataxic gait and cerebellar manifestations, followed by seizures and developmental delay. The rest of the four (18.2%) cases presented initially with delayed motor development.
In Europe, USA, and the Far east, the initial clinical presentation in cases of LINCL is mainly seizures (Mole et al., 2005), which is in agreement with our results.
All 10 of our Egyptian patients with infantile NCL presented clinically with delayed motor development, followed by seizures, which is similar to other reports (Goebel and Wisnieoiski, 2004).
Nonetheless, extensive clinical, biochemical, and molecular genetic studies should be initiated in Egypt to investigate the different main clinical presentations and genetic forms in Egyptian NCL cases.
All cases were subjected to EEG tracings, which show background slowing with high amplitude and spikes especially in the occipital region, which is similar to those in other reports (Veneselli et al., 2000). Also, ERG and VEP showed different degrees of degeneration of the retina with affection of the rods and cones. Also, increased latencies of VEP were found in our cases, which is in agreement with the results of Cooper et al. (2015).
Neuroradiological findings showed cerebellar atrophy in 28 out of our 32 (87.5%) cases among our Egyptian NCLs patients that was detected mainly in patients with LINCL.
Cortical brain atrophy could be detected in MRI of the brain in 20 out of our 32 (62.5%) NCLs cases and it was mainly in the infantile type NCL patients. This is in contrast with other reports that found that cortical brain atrophy is the main feature in the MRI of the brain of patients with NCLs (Autti et al., 1997). Also, this is in contrast with the results of Vanhanen et al. (2004), who reported that 19 out of 22 (86.36%) cases of NCL developed cortical brain atrophy irrespective of the type of NCL.
Periventricular deep white matter hyper intensity on T2-weighted images on MRI of the brain was found in 16 out of 32 (50%) Egyptian patients with NCLs. This could be attributed to loss of myelin and gliosis. Our results are similar to those in other reports (Geracts et al., 2016).
Also, hypodensity of thalami on T2-weighted images could be observed in only six out of 32 of our NCL cases (18.75%), which could be because of the deposition and accumulation of the lipofuscin material (Autti et al., 1997).
None of our cases showed any involvement either in the brain stem or in the internal capsule. This is in contrast to the result of Topcu et al. (2004), who reported that patients with the Turkish variant of LINCLs showed involvement of the brain stem and increased signal intensity in the posterior crus of the internal capsule.
Histopathological NCLs are characterized by the accumulation of autoflorescent lipofuscin pigments. This accumulated material takes different forms, for example, GRODs, curvy linear profiles, fingerprint profiles, as well as rectilinear complex. As these deposits are not disease specific and are not confined to the nervous system, they can be visualized in biopsies from more accessible tissues, for example, skin, blood lymphocytes, skeletal muscles, and conjunctival and buccal mucosa. Therefore, we used buccal mucosa through gingival biopsies for electro microscopic studies. Our results confirmed the presence of GRODs amongst our Egyptian patients. Gingival biopsy is the least expensive, noninvasive, and easily accessible outpatient technique that may be used.
By genetic analysis of our Egyptian patients, the diagnosis of 14 cases of LINCL Egyptian patients was confirmed with WES. Our results showed that eight cases had CLN6 gene mutations, which is the most common gene to be mutated affection among patients with LINCL. This is in agreement with the study carried out by Heine et al. (2004).
Amongst our cases, the main clinical presentations were developmental delay, mainly motor, and cerebellar ataxia, followed by seizures, which is similar to the study carried out by Nitta et al. (2016). However, this is in contrast with the report of Mole et al. (2005), who found that early visual failure was the main clinical presentation among cases of CLN6 gene mutations.
Also, MRI of the brain of our CLN6 gene mutation cases was characterized mainly by cerebellar atrophy in four cases, whereas concomitant cortical atrophy was observed in the other three CLN6 mutation cases. This is similar to the study of Nitta et al. (2016). Periventricular leukomalacia and basal ganglia hypodensity were observed in seven out of our eight cases with CLN6 mutations. This is in agreement with the results of Autti et al. (1997).
CLN7 gene mutations were detected in four out of our 14 cases by WES. All these case presented clinically with generalized seizures including myoclonic attacks. MRI of the brain of CLN7 cases showed cerebellar atrophy and associated periventricular leukomalacia, which is similar to other reports (Charbol et al., 2013), but none of our cases showed thinning of the corpus callosum, which is in contrast with the study carried out by Cooper et al. (2015).
Only two cases of our cohort showed CLN2 gene mutations. They presented clinically with myoclonic seizures and akinetic attacks. On MRI, these two cases showed combined cortical and cerebellar atrophy. Cerebellar atrophy affected both the hemispheres and the vermi, which is similar to the results reported by Schulz et al. (2013).
Generally, NCL should be considered clinically in any case of unexplained progressive encephalopathy associated with seizures and cerebellar atrophy on MRI of the brain.
As NCLs are autosomal recessive inherited neurodegenerative disorders, prenatal testing is possible. Thus, if there is any deficiency in the biochemical enzyme activity or involvement of any known CLN mutation gene, potential prenatal testing could be performed on the chorionic villus of fetal cells.
Understanding the biology of CLN genes and proteins, whether wild type or mutated forms, will help expand our therapeutic research. Despite expanded knowledge of the genetic background of these disorders, there has been little progress toward understanding how these mutated genes exert their adverse effects (Cooper et al., 2015).
Williams and Mole (2012) have provided experts with a modified classification for genetic, biochemical, and ultrastructural diagnostic techniques that could improve the clinical management. This modified diagnostic classification will hopefully improve scientific research and possibly targeted therapies.
Gene therapy in LINCL caused by pathogenic variants in CLN2 was initiated. While stem cell therapy for phenotypes' variants in CLN1 and CLN2 is in progress (Mole, 2014). Still, there is a clinical trial using the immuno-suppressant “Mycophenolate” in individuals with juvenile CLN3 (Worgall et al., 2008).
Therefore, expanding molecular genetic data of our Egyptian NCL cases will broaden the field of research and increase the knowledge of therapeutic approaches for the treatment of this fatal disease. 
The authors thank Dr Vincent Cantagrel, at the Imagine Institute, INRSM UMR 1163, laboratory of molecular and pathophysiological basis of cognitive disorders, 75015, Paris, France, for his support and collaboration in performing WES in our Egyptian patients to identify the affected disease-causing genes.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Autti T, Raininko R, Vanhanen SL (1997). Magnetic resonance techniques in neuronal ceroid lipofuninoses and some other lysosomal diseases affecting the brain. Curr Opin Neurol 106
Chartbol B., Cailaud C., Minussian B., (2013). Neuronal Ceroid lipofucinosis. Handb. Clin Neurol. 113
Claussen M, Hein P, Knispel J, Goebel HH, Kohlschuther A (1992). Incidence of neuronal ceroid lipofuncinosis in West Germany, variation of a method for studying autosomal recessive disorders. Am J Med Genet 42
Cooper J, Tarezyluk M, Nelvagel H (2015). Neuronal ceroid lipofucinoses. Biochim Biophys Acta 1852
Geracts R, Koch S, Hastings M (2016). Neuronal ceroid lipofucinoses. Orphan J Rare Dis 11
Goebel HH, Wisnieoiski KE (2004). Current state of clinical and morphological features in human NCL. Brain Pathol 14
Greaves J, Chamberlain LH (2007). Palmitoylation-dependentprotein sorting. J Cell Biol 176
Heine C, Koch B, Storch S, Kohlschutter A, Palmer DN, Braulke T. (2004). Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A. J Biol Chem 279
Jalanko A, Braulke T (2009). Neuronal ceroid lipofucinoses. Biochim Biophys Acta 1793
Mole SE (2014). Development of new treatments for Batten disease. Lancet Neurol 13
Mink JW, Augustin ER, Adams HR, Marshall FJ, Kwon JM. (2013). Classification and natural history of neuronal ceroid lipofuntionsis. J Child Neurol 28
Mole SE, Williams RE (2013). Neuronal ceroid-lipofuscinoses. In: Adam MP, Ardinger HH, Pagon RA, et al
., editors. GeneReviews ® (Internet)
. Seattle, WA: University of Washington, Seattle. pp. 1993–2017.
Mole SE, Williams RE, Goebel HH (2005). Correlations between and genotype, ultrastructural morphology and clinical phenotype in the neuronal ceroid lipofucinoses. Neurogenetics 6
Mole SE, Williams R, Goebel H (2011). The neuronal ceroid lipofucinoses (Batten disease)
ed. Oxford: Oxford University Press.
Nita DA, Mole SE, Minassian BA (2016). Neuronal Ceroid Lipofucinoses. Epileptic Disord
18 (suppl 2): 573-588. Review.
Oishi K, Ida H, Kurosawa KL (1999). Clinical and molecular analysis of Japanese patients with neuronal ceroid lipofuinosis. Mol Genet Metab 66
Scaioli V, Nardocci N
(2000). A pathophysiologicalogical study of neuronal ceroid lipofucinoses in 17 patients: critical review and methodological proposal. Neuro Sci 21
Schulz A, Dhar S, Rylova S, Dbaibo G, Alroy J, Hagel C, et al.
(2004). Impaired cell adhesions and apoptosis in a novel CLN9 Batten disease variant. Ann Neurol 56
Schulz A, Kohlschütter, Mink J, Simonati A, Williams R (2013). NCL diseases-clinical perspectives. Biochim Biopthys Acta 1832
Topcu M Tan H, Yalnizdu D, Usutun A, Saatci I, Aynaci M, et al
. (2004). Evaluation of 36 patients from Turkey with neumoral ceroid lipofucinosis: clinical, neuropysiological neurological and histopathoglogical studies. Turk J Pediatr 46
Uvebrant P, Hagberg B (1997). Neuronal ceroid lipofucinoses in Scandinaria, Epidemiology and Clinical Pictures. Neuropediatrics 28
Vanhanen SL, Puranel J, Autti T, Raininko R, Liewendahl K, Nikkinen P, et al
. (2004). Neuroradiological findings in infantile neuronal ceroid lipofusinoses at different stages of the disease. Neuropediatrics 35
Vantianen SL, Sainio K, Lappi M, SantavuoriP(1997). EEG and evoked potentials in infantile ceroid lipofucinoses Dis Med Child Neurol 39
Veneselli E, Biancheri R, Perrone MV, Buoni S, Fois A (2000). Neuroceroid lipofucinoses clinical and ECG findings in a large study of Italian cases. Neurol Sci 21
Voznyi YV, Keulemans JL, Mancini GM, Catsman-Berrevoets CE, Young E, Winchester B, et al
. (1999). A new simple enzyme assay for pre-and postnatal diagnosis of infantile neuronal lipofuscinosis (INCL) and its variants. J Med Genet 36
Williams RE, Mole SE (2012). New nomenclature and classification scheme for the neuronal ceroid lipofuscinoses. Neurology 79
Worgall S, Sondhi D, Hackett NR, Kosofsky B, Kekatpure MV, Neyzi N, et al
. (2008). Treatment of late infantile neuronal ceroid lipofucinosis by CNS administration of a serotype adeno-associated virus expressing CLN2. Hum Gene Ther 19
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]