Classification of the neuronal ceroid-lipofuscinoses: Expansion of the atypical forms

The neuronal ceroid-lipofuscinoses (NCL) are a group of different genetic diseases. The major types of NCL are expressed by six forms which represent different clinicopathologic and genetic forms. These are CLN-1, Infantile; CLN-2, Late Infantile; CLN-3, Juvenile; CLN-4, Adult-Recessive; CLN-5, Adult-Dominant; and CLN-6, Early Juvenile. The distinction between CLN-4 and CLN-5 is still disputatious. CLN-6 has been called CLN-5. A seventh classification of NCL represents from 12 to 20% of those afflicted. This group consists of an extensive array of atypical types of ceroid-lipofuscin accumulation in the secondary lysosomes of neurons and cells of other tissues (e.g., skin, conjunctiva, and lymphocytes) or by presumed clinical and genetic relationships. The authors have identified 15 atypical subtypes of NCL. These as a group are here described as a seventh form. Further biochemical, molecular, and genetic studies will identify more precisely the phenotypic and genotypic expression of these “minor” forms of NCL. © 1995 Wiley-Liss, Inc.     

Abnormal acid phosphatases in neuronal ceroid-lipofuscinoses

Acid phosphatases in brain and cultured lymphoblasts from patients affected with neuronal ceroid-lipofuscinoses (NCL) were studied by starch gel electrophoresis. After electrophoresis the gel was incubated with 4-methyl umbelliferyl phosphate at pH 4.5 and the fluorescent reaction product was visualized under ultraviolet light. Control brain showed a single band with mobility of about 1 cm while NCL patients showed two additional fast moving bands. In the lateinfantile, and in the adult form (Kufs disease), the middle band was prominent while the fast moving band was predominant in juvenile NCL. In long-term lymphoblasts, controls showed a single band of acid phosphatase activity while both juvenile and late-infantile NCL showed two additional fast moving bands. Obligate heterozygotes showed reduced levels of the fast moving bands. Fluorometric assay of acid phosphatase using 4-methylumbelliferyl phosphate as substrate showed a 2-fold increase in activity in the patients. The increased acid phosphatase activity is completely inhibited by tartrate. Lymphocyte hexosamnidase activities were unchanged in NCL patients lymphoblasts. Studies on brains of NCL patients and on cultured lymphoblasts from families with late-infantile and juvenile form of NCL showed that abnormal acid phosphatase is characteristic of NCL © 1995 Wiley-Liss, Inc.     

Incidence of neuronal ceroid-lipofuscinoses in West Germany: Variation of a method for studying autosomal recessive disorders

The incidence of neuronal ceroid-lipofuscinoses (NCL) in West Germany was determined using a novel method which is applicable to other autosomal recessively inherited diseases. Questionnaires were sent to all pediatric departments (answer rate 189/276, 68%), schools for the blind (39/46, 85%), and neuropathological institutes (15/22,68%). Diagnoses were accepted only when based on firm clinical and/or electron microscopic criteria; 207 such identified patients were sorted according to year of birth. Plotting the cumulative number of new cases per year against the year of birth resulted in a slightly S-shaped curve. Before the year 1962, the curve is relatively flat, probably due to inefficient case registration. Between 1968 and 1977, the slope of the curve is constant–a steep, nearly straight line. Thereafter the curve flattens out again, likely due to inefficient registration of young, still undiagnosed patients. We interpret the central segment of the curve, which is continuously straight over a period of 10 years and corresponds to 92 patients, as a period in which efficient registration of new cases occurred. The number of live births being 7,211,543 during the same period, the NCL incidence is calculated to be 1.28 per 100,000 live births (0.71 for juvenile NCL and 0.46 for late infantile NCL).     

Neuronal ceroid-lipofuscinoses in Italy: An epidemiological study

To establish the incidence of neuronal ceroid-lipofuscinoses (NCL) in Italy, we sent a questionnaire to all Neuropediatric and Child Neuropsychiatric Departments (answer rate 15/34 = 44%). Diagnoses were accepted only when based on firm clinical and/or electron microscopic criteria. We collected 58 cases born between 1966–1991 (2 infantile NCL, 37 late infantile NCL, and 19 juvenile NCL). The incidence was calculated only on patients born between 1974–1984. In this period, the incidence of overall NCL in the Italian population was calculated to be 0.56 per 100,000 live births (0.36 for late infantile NCL, and 0.20 for juvenile NCL). Our data show that infantile NCL is very rare in Italy, and that late infantile seems to be the most frequent form of NCL. © 1995 Wiley-Liss, Inc.     

Linkage analysis in juvenile neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinosis (NCL, Batten disease) is an autosomal recessive disease characterized by progressive mental retardation, cortical atrophy, seizures, and retinal degeneration. Several subtypes have been delineated on the basis of age-at-onset and histological characteristics; the most common is the juvenile (JNCL) form. Recently, the gene for JNCL was shown to reside on chromosome 16 through linkage studies to the haptoglobin locus and anonymous DNA markers using numerous European families. We have now examined 8 families from North America with JNCL for linkage to markers in 16q21–23. Results in 3 families tend to support linkage to chromosome 16; 3 families remained uninformative, and 2 families produced negative lod scores in this region. A test of homogeneity was suggestive, but could not significantly reject the null hypothesis of homogeneity. We are continuing to collect families, particularly those with multiple living affecteds, and are identifying other probes in this region. Given close localization on chromosome 16 for JNCL, molecular strategies, including candidate gene strategies, are being explored.     

Linkage analysis of late-infantile neuronal ceroid-lipofuscinosis

The neuronal ceroid-lipofuscinoses (NCL) are a group of neurodegenerative disorders with an autosomal-recessive pattern of inheritance. There are 3 main categories of childhood NCL, namely, infantile, late-infantile, and juvenile NCL. These can be distinguished on the basis of age of onset, clinical course, and histopathology. A number of variant forms of NCL have also been described, and these show symptoms intermediary between the main classical forms. The genes for both the infantile and juvenile forms of NCL have previously been mapped to chromosome areas 1p32 and 16p12, respectively. The gene for late-infantile NCL (LINCL), CLN2, has been excluded from both these loci, but its location is as yet unknown. Recently, CLN5, the gene for the Finnish variant form of LINCL, was mapped to 13q21.1–32. Using the 3 microsatellite markers which were most tightly linked to CLN5, we have excluded CLN2 from this region using a subset of 17 families. Thus, CLN2 represents a fourth distinct genetic locus involved in the pathogenesis of NCL. © 1995 Wiley-Liss, Inc.     

New manifestations in an infant with Neu Laxova syndrome

The neuronal ceroid-lipofuscinoses (NCL), also known as Batten disease, are a not uncommon group of disorders affecting infants, children, and young adults. The abnormal ultrastructural profiles seen in NCL are used for standard diagnosis; however, they can be missed, and are also found in other neurodegenerative conditions. Furthermore, there is an overlap between the types of inclusion profiles among the different forms of NCL. Therefore, a more specific and biochemically-based marker is necessary to confirm the diagnosis of NCL. Antibodies raised against the storage material from the ovine form of NCL (mitochondrial ATP synthase subunit c) were utilized to determine whether NCL could be distinguished from other metabolic-neurodegenerative disorders. By immunoblotting and immunohistochemistry, several brain samples of well-evaluated NCL cases confirmed increased accumulations in all NCL cases except in the brain of an infantile-onset NCL patient. The immunoblot studies of skin fibroblasts and brain were sensitive but not highly specific to NCL, due to the recognition of this material in normal controls as well as in other neurogenetic diseases. Immunocytochemistry of skin fibroblasts clearly distinguished LINCL and JNCL cases from controls, and with further refinement has the potential for becoming a diagnostic tool. © 1995 Wiley-Liss, Inc.     

Retention of lysosomal protein CLN5 in the endoplasmic reticulum causes neuronal ceroid lipofuscinosis in Asian Sibship

The neuronal ceroid lipofuscinoses (NCLs) form a group of autosomal recessively inherited neurodegenerative disorders that mainly affect children. Ten NCL forms can be distinguished by age at onset, clinicopathologic features, and genetics. In eight of these forms, the underlying genes have been identified. At present, approximately 10% of all patients do not fall into one of the eight known genetic forms of NCL. We have identified two Asian families with two novel homozygous mutations in the CLN5 gene. In the first Pakistani family, two children developed symptoms of an early juvenile NCL. After exclusion of mutations in genes known to be associated with this age of onset in families from many different countries (CLN1, CLN2, CLN3, CLN6, CLN8 and CLN10) SNP array‐based homozygosity mapping led to the identification of a novel homozygous mutation c.1072_1073delTT (p.Leu358AlafsX4) in CLN5. In the second Afghan family, two children developed symptoms of a late infantile NCL. The mutation c.1137G>T (p.Trp379Cys) in CLN5 was identified. The affected children in these families represent the first reported CLN5 patients originating in Asian sibships. Expression analysis showed that mutant p.Leu358AlafsX4 CLN5 is truncated and lacks a used N‐glycosylation site at Asn401. The missense mutation p.Trp379Cys affected neither the size nor glycosylation of the CLN5 protein. Double immunofluorescence microscopy showed that while the wild‐type CLN5 protein is localized in lysosomes, both mutant CLN5 proteins are retained in the endoplasmic reticulum rather than reaching the lysosome. © 2009 Wiley‐Liss, Inc.     

Application of chromosome 16 markers in the differential diagnosis of neuronal ceroid-lipofuscinosis

Accurate diagnosis of neuronal ceroid lipofuscinosis (NCL) is important for a correct prognosis of the disease and for genetic counseling. Up to now, no direct diagnostic test has been available for NCL. The clinical diagnosis is made on the basis of symptoms, neurophysiological, neuroradiological, and specific lipopigment pattern data. Recent advances in the genetics of NCL have enabled us to use polymorphic DNA markers linked to the CLN1 and CLN3 loci as a tool in the differential diagnosis of NCL. We have applied genetic analysis with polymorphic DNA markers flanking the CLN3 gene on chromosome 16 to two consanguineous families in which NCL occurs. In the first family, which is of Turkish extraction, two patients suffering from a protracted form of juvenile NCL previously had been diagnosed with juvenile NCL. Haplotypes from this family indicate that the patients and their healthy sibling are haplo-identical, suggesting that this protracted form of juvenile NCL is not linked to the CLN3 locus. In the second family, which is of Moroccan origin, one patient suffers from the early juvenile variant of NCL (Lake-Cavanagh). In this family, the patient and one of the healthy siblings have identical haplotypes, excluding linkage of early juvenile NCL to the CLN3 locus on 16p12.1-11.2. Therefore, these cases from different populations demonstrate that haplotype analysis can be used as an additional method to exclude the diagnosis of juvenile NCL. © 1995 Wiley-Liss, Inc.     

Report on the fifth international conference on neuronal ceroid-lipofuscinoses

Distal arthrogryposis IIB is characterized by contractures of the distal joints (especially of the fingers and toes) and ptosis. We recently encountered a father and son with these manifestations. The father was reported 54 years ago as a case of amyoplasia congenita (arthrogryposis multiplex congenita). Both father and son have distal joint contractures, most severe in the hands and feet, as well as ptosis and ophthalmoplegia. In addition, these patients have an unusual distribution of hair loss, and conical teeth. Whether these latter findings are related to the type of distal arthrogryposis present in this family is not known. In spite of their physical limitations both father and son have maintained an active life-style. © 1995 Wiley-Liss, Inc.     

Spectrum of CLN6 mutations in variant late infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative diseases of childhood. CLN6, the gene mutated in variant late infantile NCL (vLINCL), was recently cloned. We report the identification of eight further mutations in CLN6 making a total of 18 reported mutations. These mutations include missense, nonsense, small deletions or insertions, and two splice-site mutations. Ten mutations affect single amino acids, all of which are conserved across vertebrate species. Minor differences in the pattern of disease symptom evolution can be identified. One patient with a more protracted disease progression was a compound heterozygote for a missense mutation and an unidentified mutation. Fifteen CLN6 mutations occur in one or two families only, and families from the same country do not all share the same mutation. Unlike NCLs caused by mutations in CLN1, CLN3, CLN5, and CLN8, there is no major founder mutation in CLN6. However, one mutation (E72X) is significantly more common in patients from Costa Rica than two other mutations present in that same population. In addition, a 1-bp insertion (c.316insC) is associated with families from Pakistan and I154del may be common in Portugal. A group of Roma Gypsy families from the Czech Republic share two disease-associated haplotypes, one of which is also present in a Pakistani family, consistent with the proposed migration of the Roma from the Indian subcontinent 1,000 years ago. All mutations are recorded in the NCL Mutation Database together with their country of origin for use in the development of rapid screening assays to confirm diagnosis and to facilitate carrier testing appropriate to a population.     

Novel mutations in the CLN6 gene causing a variant late infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of autosomal recessive neurodegenerative diseases comprising Batten and other related diseases plus numerous variants. They are characterized by progressive neuronal cell death. The CLN6 gene was recently identified, mutations in which cause one of the variant late infantile forms of NCL (vLINCL). We describe four novel mutations in the CLN6 gene. This brings the total number of CLN6 mutations known to 11 in 38 families. This suggests that the CLN6 gene may be highly mutable. An American patient of Irish/French/Native American origin was heterozygous for a 4-bp insertion (c.267_268insAACG) in exon 3. The other allele had a point mutation (c.898T>C) in exon 7 resulting in a W300R amino acid change. Two Trinidadian siblings of Indian origin were homozygous for a mutation at the 5' donor splice site of exon 4 (IVS4+1G>T), affecting the first base of the invariant GT at the beginning of intron 4. The fourth novel mutation, a double deletion of 4 bp and 1 bp in exon 7 (c.829_832delGTCG;c.837delG), was identified in a Portuguese patient heterozygous for the I154del Portuguese CLN6 mutation. Four of the 11 mutations identified are in exon 4. Three Portuguese patients with clinical profiles similar to CLN6 patients without defects in CLN6 or other known NCL genes are described. We conclude the following: 1) the CLN6 gene may be a highly mutable gene; 2) exon 4 must code for a segment of the protein crucial for function; 3) vLINCL disease in Portugal is genetically heterogeneous; 4) the I154del accounts for 81.25% of affected CLN6 Portuguese alleles; and 5) three vLINCL Portuguese patients may have defects in a new NCL gene.     

Neurology of the neuronal ceroid-lipofuscinoses: Late infantile and juvenile types

My experience with more than 80 cases of the late infantile and juvenile forms of the neuronal ceroid-lipofuscinoses over the last 5 years has led to the following realizations. The 2 variants are neurologically distinct entities and probably are the result of different genetic defects. Treatment includes supportive measures and anticonvulsant medication. Therapy for behavioral and psychiatric disturbances in the juvenile type proves to be particularly challenging as neuroleptic medications tend to worsen parkinsonian like symptoms. Neuropathologic and neuro radiologic explanation of clinical symptomatology correlates best with neuronal loss and not neuronal storage. There is a paucity of neuropathologic documentation of these 2 types; additional reports are encouraged.     

Evidence for lipase abnormality: High levels of free and triacylglycerol forms of unsaturated fatty acids in neuronal ceroid-lipofuscinosis tissue

Total lipid obtained from normal and different forms of neuronal ceroid-lipofuscinoses (NCL) tissues was analyzed by high performance thin layer chromatography (HPTLC). We observed a large (>6-fold) increase in a lipid band corresponding to triolein for NCL dog pancreas and spleen and juvenile human NCL brain and infantile NCL spleen. The accumulation was less pronounced for the brain samples but apart from increased dolicholmonophosphate levels, other lipids appeared normal. Normal dog, goat, or human spleen contained virtually no triacylglycerol, and of the pathological controls, β-mannosidosis goat spleen showed no triacylglycerol band at all. A sample of human spleen from a patient with lymphoma-associated splenomegaly displayed a strong triacylglycerol band, but gas chromatography-mass spectrometry (GC/MS) of the bands showed an equal increase in both saturated and unsaturated fatty acid containing triacylglycerols in the splenomegaly sample, in keeping with the notion of non-specific fat deposition in damaged tissue. In contrast, in all the NCL samples (spleen, pancreas, and brain) a prominent increase in the proportion of unsaturated fatty acids was observed in both free fatty acid and/or triacylglycerol bands following GC/MS. The NCL-English setter dog pancreas showed a major presence of oleic acid (18:1) (twofold increase) as compared to normal, while dog and infantile human NCL spleen samples and juvenile Batten brain (human) displayed a robust increase in linoleic acid (18:2) and sometimes in oleic acid and arachidonic acid (20:4) (for infantile human NCL spleen). For the infantile human NCL spleen sample an increase in linoleic acid in both free fatty acid (3.2-fold) and triacylglycerol (10-fold) was observed. This suggests a new mechanism by which polyunsaturated fatty acids can be transported to various parts of an NCL-affected mammalian body in packages such as triacylglycerol and/or serum albumin:free fatty acid complex. Such polyunsaturated fatty acids could eventually lead to the ceroid-lipofuscin pigment characteristic of NCl tissues through peroxidation to aldehyde with subsequent Schiff base formation. This could possibly explain the deposition of ceroid-lipofuscin in widely different and non-proximal tissues such as the pancreas and the brain.     

Linkage map of the chromosomal region surrounding the infantile neuronal ceroid lipofuscinosis on 1p

The neuronal ceroid lipofuscinoses (NCLs) of childhood are divided into 3 main types according to age-of-onset, clinical course, and neurophysiological and neuropathological findings: infantile, late infantile, and juvenile. All forms are inherited as an autosomal recessive trait, and their biochemical background is still unknown. The infantile type (INCL) with the earliest age-of-onset and the most severe clinical course, occurs in Finland with an incidence of 1:20,000, i.e., 116 patients have been found in our country up to now, whereas only about 50 cases have been reported from other parts of the world. Earlier we reported the linkage of INCL to the short arm of chromosome 1. Here we describe a more precise linkage map of this area. Our current map places the INCL mutation between D1S57 and D1S79; D1S7 has so far shown no recombination events between the marker and the disease (lod score 4.55 at θ = 0.00). Our material includes 64% of all living patients in Finland, and no linkage disequilibrium of haplotypes is seen, using the 2 physically close markers D1S57 and D1S79. This finding as well as our LINKMAP analyses suggest that the distance between the disease locus and the flanking markers is about 3–4 cm.     

Genetic heterogeneity of neuronal ceroid lipofuscinosis in The Netherlands

An overview of patients in the Netherlands who are known to us with neuronal ceroid lipofuscinosis (NCL) is presented. Several CLN genes involved in NCL have been isolated or mapped. We have analyzed families with different types of NCL with polymorphic markers linked to CLN loci to investigate the genetic heterogeneity of NCL in the Netherlands. Haplotype analysis suggests that in addition to the CLN2 and CLN6 genes another gene is involved in at least one family with late infantile NCL in the Netherlands. The CLN2 and CLN6 loci have also been excluded in a family with protracted juvenile NCL.     

Comparative biology of the neuronal ceroid-lipofuscinoses (NCL): An overview

Multiple forms of ceroid-lipofuscinosis occur in human beings and animals. They are characterized by brain and retinal atrophy associated with selective necrosis of neurons. This neurodegenerative disease appears associated with the disease process rather than storage of fluorescent lipopigment per se, and there is now growing evidence that pathogenesis may involve mitochondria rather than a primary defect of lysosomal catabolism. Of the forms of ceroid-lipofuscinosis studied, most but not all reflect accumulation of subunit c of mitochondrial ATP synthase. If there is a common denominator between all forms other than the presence of fluorescent lipopigment, then it may be the accumulation of hydrophobic protein. Analogous diseases in animals can be expected to reflect the same spectrum of biochemical changes, and they warrant in-deptb study to help understand the pathogenesis and heterogeneity of the group. © 1995 Wiley-Liss, Inc.     

神经元蜡样脂褐质沉积病5例的脑电与临床表现

目的：对5例DNA测序证实的神经元蜡样脂褐质沉积病（neuronal ceroid lipofuscinosis,NCL）患儿的临床和视频脑电图（V—EEG）进行分析,探索临床和EEG的对应关系。方法：对5例NCL患儿进行详细病史询问。神经学查体,行常规VEEG2h监测,观察临床特点和EEG的对应关系。结果：本组5例NCL患儿中,发现在1例病情较轻的患儿EEG表现为局限性慢波和慢波后尖慢波,视频未见抽搐发作;病情重的4例（肌力≤Ⅱ级）患儿中．3例有抽搐发作（1例有侧别优势,其对侧额区周期性痫样放电明显）,其中1例在EEG低平的状态下抽搐,2例的周期性痫样放电与抽搐无固定锁时关系;另1例未见明显抽搐（无任何动作）。结论：EEG中特征性的周期性痫样放电发生于NCL病情重的患儿,这些患儿中有的甚至出现头皮EEG低平活动。     

Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease)

The ceroid-lipofuscinoses (Batten disease) are neurodegenerative inherited lysosomal storage diseases of children and animals. A common finding is the occurrence of fluorescent storage bodies (lipopigment) in cells. These have been isolated from tissues of affected sheep. Direct protein sequencing established that the major component is identical to the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mitochondrial ATP synthase and that this protein accounts for at least 50% of the storage body mass. No other mitochondrial components are stored. Direct sequencing of storage bodies isolated from tissues of children with juvenile and late infantile ceroid-lipofuscinosis established that they also contain large amounts of complete and normal subunit c. It is also stored in the disease in cattle and dogs but is not present in storage bodies from the human infantile form. Subunit c is normally found as part of the mitochondrial ATP synthase complex and accounts for 2–4% of the inner mitochondrial membrane protein. Mitochondria from affected sheep contain normal amounts of this protein. The P1 and P2 genes that code for it are normal as are mRNA levels. Oxidative phosphorylation is also normal. These findings suggest that ovine ceroid-lipofuscinosis is caused by a specific failure in the degradation of subunit c after its normal inclusion into mitochondria, and its consequent abnormal accumulation in lysosomes. This implies a unique pathway for subunit c degradation. It is probable that the human late infantile and juvenile diseases and the disease in cattle and dogs involve lesions in the same pathway.     

A successful approach for the detection of Fabry patients in Argentina

Fabry disease is an X-linked lysosomal disorder caused by the deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). In males, the laboratory diagnosis is based on the demonstration of decreased levels of alpha-Gal A activity, while in females, the disease is diagnosed by the identification of a mutation in alpha-Gal A gene. Fabry disease in Argentina is underdiagnosed. To date, no comprehensive screening study of Fabry disease in our country has been reported. The present study aimed at developing a targeted screening for the detection of Fabry patients from Argentina based on the set of typical signs and symptoms. We received 121 blood samples from probable Fabry patients for enzymatic and genetic assay. We diagnosed six Fabry patients from six unrelated families, representing a yield of detection of 4.96%. The mutations detected in five of the families analysed were missense mutations: p.Leu243Trp, p.Asp155His, p.Leu415Pro, p.Cys94Tyr and p.Leu191Pro. After the detection of a Fabry patient, his/her relatives were also screened. In the course of these family studies, other 64 Fabry patients, 29 males and 35 females, were detected. To our knowledge, this is the first comprehensive screening of Fabry disease in Argentina. We detected 70 patients in a period of 2.5 years. The development of targeted protocols and the constitution of interdisciplinary groups for the identification of patients with Fabry disease are recommended to obtain a higher yield in the process.