Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Depletion of alcohol (hexanol) dehydrogenase activity in the epidermis and jejunal mucosa in Sjögren-Larsson syndrome.

Using a histochemical technique, we have demonstrated a consistent deficiency of alcohol (hexanol) dehydrogenase activity within the epidermis and jejunal mucosa of patients with Sjögren-Larsson syndrome. Biochemical assay of the fatty alcohol: NAD oxidoreductase activity in cultured fibroblasts and leukocytes from these patients showed deficient activities compared with controls. The histochemical and biochemical results are complementary, and the simpler histochemical method can be used reliably for initial screening of patients with ichthyosis in whom a diagnosis of Sjögren-Larsson syndrome is suspected.

Prenatal diagnosis of lysosomal storage diseases.

The prenatal diagnosis of lysosomal storage disorders can be achieved, once the diagnosis is confirmed in the index case, by a variety of techniques including analysis of amniotic fluid, asay of enzymic activity in cultured amniotic fluid cells, cultured chorionic villus cells and by direct assay of activity in chorionic villus samples. These studies can be accompanied by ultrastructural observations which give an independent means of diagnosis. In some instances molecular genetic studies for mutation detection or linkage analysis are appropriate for prenatal diagnosis. Pseudodeficiencies of some of the lysosomal enzymes, which cause no clinical problems, can complicate the initial diagnosis particularly in metachromatic leucodystrophy where the pseudodeficiency is more common than the disease itself. Mutation analysis as well as enzyme assay is necessary not only in the index case but also in the parents before the same techniques are applied to a sample for prenatal diagnosis. A large number of lysosomal storage disorders may present as fetal hydrops and the diagnosis can be established at this late stage by fetal blood sampling and examination by microscopy as well as by biochemical assay of the appropriate enzyme or metabolite in amniotic fluid. All prenatal diagnoses in which an affected fetus is indicated should have confirmation of the diagnosis as soon as possible to reassure anxious parents, and to act as audit of the laboratory's competence to undertake prenatal diagnosis. A combined approach to prenatal diagnosis involving biochemical, molecular genetic and morphological studies is recommended.

Adult Niemann-Pick disease: its relationship to the syndrome of the sea-blue histiocyte.

Three unrelated female patients with adult Niemann-Pick disease are described. All the patients had reduced coagulation factors and involvement of the marrow, liver, spleen and lungs. Two patients were shown to have abnormal platelet function; two patients also had pingueculas and a late onset of a menarche. Foam cells and sea-blue histiocytes were seen in the marrow and livers in all three patients, in the spleen in two patients in the lymph nodes in one patient. The clinical presentation, the histologic appearance, the histochemical staining reactions, the lipid analysis and the ultrastructure were all consistent with a diagnosis of adult Niemann-Pick disease. On the basis of these observations, it is clear that adult Niemann-Pick disease is a cause of the syndrome of the sea-blue histiocyte. The existence of the syndrome of the sea-blue histiocyte as a separate entity is also questioned.

Infantile systemic hyalinosis: newly recognized disorder of collagen?

Four infants with stiff skin and painful joint contractures in the first few months of life are described. Other features included small papules, particularly on the face and trunk, perianal nodules, hyperpigmentation over the metacarpophalangeal joints and over the malleoli, gingival hyperplasia, persistent diarrhea, and failure to thrive. Two of these infants died before the age of 18 months. In each case hyaline material was found in the papillary dermis. Ultrastructurally, there was a distinctive fibrillogranular appearance in which a banding pattern could be observed. This material was also found within membrane-bound vacuoles in macrophages and fibroblasts. It had an appearance and localization identical with that of collagen type VI. These features are similar to those reported in juvenile hyaline fibromatosis. It is believed that these infants have a closely related, but nonetheless distinctive, inherited disorder of collagen.

Tissue and cellular distribution of subunit c of ATP synthase in Batten disease (neuronal ceroid-lipofuscinosis).

The major protein component of the storage bodies in the late infantile (LIB) and juvenile (JB) forms of Batten diseases is subunit c of ATP synthase (subunit c). Ultrastructurally the stored material may appear as curvilinear bodies, fingerprint profiles, or a mixture of both, dependent upon the form of Batten disease and the cell type. The mnd/mnd mouse, an animal model for Batten disease, also stores subunit c and has loosely stacked lamellae within the neurons of the brain and in other cells and tissues. Using a range of tissue samples, immunolocalization, using avidin-biotin techniques at the LM level and postembedding immunogold-labelling (5 nm) with silver enhancement at the EM level, were used to investigate specific subunit c immunoreactivity. Subunit c storage was displayed in a number of cells, including neurons, muscle cells, adipocytes, macrophages, endothelial and some epithelial cells, and exocrine and endocrine cells. By EM, subunit c was localized to all curvilinear-type storage bodies, but to nowhere else within the cell. It was not present over fingerprint profiles, the characteristic storage pattern of neurons within the JB gut, possibly due to steric factors. Preliminary studies in the mnd mouse showed subunit c immunoreactivity localized to storage profiles seen ultrastructurally in neurons of the brain, and liver and heart cells. We suggest that accumulation and distribution of subunit c within a variety of cell types, and its consistent absence in others, may be related to the particular cell type's longevity and its metabolic demand.

Late-infantile Batten disease: purification of the subunit c of the mitochondrial ATP synthase from storage material.

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 x 10(6) Da; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing < 1% lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies.

Bone marrow transplantation in Batten disease (neuronal ceroid-lipofuscinosis). Will it work? Preliminary studies on coculture experiments and on bone marrow transplant in late infantile Batten disease.

Lymphocytes from a patient with preclinical late infantile Batten disease were cultured alone and with lymphocytes from donors, and the fate of the curvilinear inclusions characteristic of the disease was monitored by electron microscopy. There was no evidence of transfer of deficient enzyme or factor that might have caused removal of the stored material, and the curvilinear profiles remained in the cultured cells without signs of degradation. Cells stimulated to divide with phytohaemaglutinin did not exhibit storage in culture suggesting that storage is a function of the age of the cell. The patient received a bone marrow transplant at 2 7/12 years while still clinically unaffected, and the effect on lymphocytes and cells in skin and rectal biopsies was monitored by electron microscopy over a period of 9 months until the donor marrow became displaced by the host cells. He has had one seizure and now has neurophysiological evidence of late infantile Batten's disease. Bone marrow transplant may have no effect on material already stored but might prevent further build-up and halt the onset of the clinical symptoms although very recent studies on early (fetal) transplants in sheep with a form of Batten disease have shown no benefit.

Stored dolichyl pyrophosphoryl oligosaccharides in Batten disease.

Each of the 3 childhood forms of Batten disease, juvenile (JB), late-infantile (LIB), and infantile (IB), have abnormally high brain concentrations of dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). In this study, the carbohydrate portions of Dol-PP-OS were analysed: in JB and LIB, they range in size from Man2GlcNAc2 to Glc3Man9GlcNAc2, predominant components being Man5-7GlcNAc2 and Glc3Man7GlcNAc2. In IB, they range from Man6-9GlcNAc2, no glucose containing oligosaccharides being identified. In Batten disease, the main subcellular location of Dol-PP-OS is within storage material, where it represents up to 7% of the dry weight. [3H]-Mannose incorporation experiments with cultured fibroblasts show that synthesis of Dol-PP-OS in JB is normal. We infer that the glycosylation intermediate Glc3Man9GlcNAc2-PP-dolichol is synthesised normally within the endoplasmic reticulum in Batten disease, but that catabolic derivatives accumulate within the lysosomes. It is unclear whether this process is central to the pathogenesis of the disease, though in IB a defect in the release of mannose residues from Dol-PP-OS is a distinct possibility.

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.

Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry.

A technique using alkaline phosphatase histochemistry on routine sections of four jejunal biopsy specimens and one necropsy sample was applied to show that alkaline phosphatase activity, normally present in the brush border, occurs in the enterocytes of patients with microvillus inclusion disease. Sections were cut at 5 micron, mounted on to glass slides, and dried overnight at 37 degrees C before staining for alkaline phosphatase activity by the indoxyl phosphatase nitro blue tetrazolium method. Incubation periods amounted to 10 minutes for biopsy specimens and 30 minutes to one hour for necropsy samples. The demonstration of alkaline phosphatase activity in routinely processed biopsy specimens provides an effective, quick, and definitive test in the diagnosis of microvillus inclusion disease without recourse to electron microscopy.

Histochemical detection of the enzyme deficiency in blood films in Wolman's disease.

The clinical diagnosis of Wolman's disease (acid esterase deficiency) can be confirmed by a histochemical staining method using blood films. Acid esterase activity is found normally in lymphocytes, but in Wolman's disease only a very low level of activity can be detected. An intermediate level of activity was demonstrated in heterozygotes. The method may also be applicable in the prenatal detection of the disease.

Cystinosis: electron microscopic evidence of lysosomal storage of cystine in lymph node.

Electron microscopic examination of a biopsy specimen of cystinotic lymph node demonstrated that cystine crystals were entirely intracellular, and that the majority, including those of the largest size, were enclosed by intact limiting membranes. Acid phosphatase activity was localized specifically at the periphery of the crystal profiles and the smallest crystal aggregates were contained in osmophilic dense bodies which had the appearance of progressively swollen lysosomes. The implications of the suggested lysosomal distribution of cystine are discussed.

Early-juvenile Batten's disease--a recognisable sub-group distinct from other forms of Batten's disease. Analysis of 5 patients.

In most cases where rectal biopsy is performed to diagnose Batten's disease, there is good correlation between biopsy appearance, age of onset, clinical course and electrophysiological parameters. As a result 3 forms of the disease have been recognised; infantile, late infantile and juvenile. In a review of rectal biopsy in Batten's disease at the Hospital for Sick Children, Great Ormond Street, we have studied the few cases in which no such correlation appeared to exist. In 5 the features are sufficiently similar to suggest a further recognisable sub-group which could be descriptively called "early juvenile". The clinical course, electrophysiological features and the absence of vacuolated lymphocytes in this subgroup are as found in the late infantile form, whereas the biopsy findings are identical to those of the juvenile form. By analogy with some of the mucopolysaccharidoses we speculate that the genes of the late infantile and juvenile forms of Batten's disease are allelic and that the "early juvenile" sub-group is a genetic compound presenting as an intermediate phenotype.

Zebra body myopathy. Clinical, histochemical and ultrastructural studies.

Clinical, electrophysiological and histological evidence is presented of a myopathy not previously described. The patient, aged 15 years, was generally weak and wasted. His condition probably began in utero, but abnormalities of the serum CPK and EMG did not become evident until adolescence. Wide variation in fibre diameter, vacuolation, calcification, increased endomysial connective tissue and intra-fibre splitting were prominent in muscle biopsy sections. Electron microscopy showed numerous rod bodies, fibre disorganisation with loss of Z-bands and occasional honeycomb structures. Frequent zebra bodies were present. Because of the large number of zebra bodies seen, the name "zebra body myopathy" is provisionally proposed.

Hirschsprung's disease: an appraisal of histochemically demonstrated acetylcholinesterase activity in suction rectal biopsy specimens as an aid to diagnosis.

Suction rectal biopsy specimens, from a series of 168 infants and children with constipation and other gastrointestinal problems, were stained with a sensitive acetylcholinesterase method, and the results were compared with routine histology, radiology, anorectal manometry, and the final diagnosis. In all cases of Hirschsprung's disease, there was an increase in numbers and sizes of cholinergic nerves in the lamina propria and muscularis mucosae. No false-positive or false-negative results were found. The test was found to be more reliable and consistent than other methods available.

Glycoconjugates in storage cytosomes from ceroid-lipofuscinosis (Batten's disease) and in lipofuscin from old-age brain.

The ceroid-lipofuscinoses (CL) are a group of inherited diseases characterised by the accumulation, in brain, of autofluorescent storage cytosomes which have similar histochemical staining properties to lipofuscin, the neuronal wear and tear pigment of old-age brain. The storage cytosomes stain strongly with periodic acid-Schiff reagent (PAS), indicating the presence of carbohydrate. In brain from each childhood form of CL, concentrations of phosphorylated dolichol (Dol-P) are 10- to 20- fold higher than in age-matched controls. Brain Dol-P concentrations are also increased between 2 and 5- fold in several different lipidoses and in elderly subjects. Much of the Dol-P which accumulates is located within the storage cytosomes. Dol-P constitutes 2-3% of the dry weight of storage cytosomes from juvenile and late-infantile CL, and 0.3-0.7% of storage cytosomes from infantile CL, ovine CL and of lipofuscin isolated from old age brain. The bulk of the Dol-P in CL brain and in isolated storage cytosomes is present as dolichyl pyrophosphoryl oligosaccharides (Dol-PP-OS). The constitutions of the oligosaccharide moieties differ in the various forms of the disease. Histochemical analysis of frozen sections of unfixed brain after extraction by various lipid solvents indicates that the major part of the PAS positive intraneuronal material in CL brain and in old-age brain has the extraction properties of Dol-PP-OS. Carbohydrate represents 4-7% of the dry weight of CL storage cytosomes and of lipofuscin. The major monosaccharide components are mannose, N-acetyl glucosamine, glucose and galactose. Depending on the form of the disease studied, up to 40% of this material can be accounted for by Dol-PP-OS. Polyacrylamide gel electrophoresis of storage cytosomes followed by lectin blotting demonstrates several low molecular weight components which bind concanavalin A. These do not coelute with the major protein components and may well be Dol-PP-OS. We conclude that Dol-PP-OS are concentrated in storage cytosomes in CL and are one of their major glycoconjugate components.

Lysosomal storage of the DCCD reactive proteolipid subunit of mitochondrial ATP synthase in human and ovine ceroid lipofuscinoses.

The ceroid lipofuscinoses (Batten's disease) are a group of neuro-degenerative lysosomal storage diseases of children and animals that are recessively inherited. In the diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. The material stored in the cells of sheep affected with ceroid lipofuscinosis is two-thirds protein. The stored material does not arise from lipid peroxidation or a defect in lipid metabolism, and the lipid content is consistent with a lysosomal origin for the storage bodies. The major protein stains poorly with Coomassie blue dye and is soluble in organic solvents. It has an apparent molecular weight of 3,500 and its amino acids sequence is identical to that of the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mammalian mitochondrial ATP synthases. Apart from removal of mitochondrial import sequences, it has not been modified post-translationally. At least 50% of the mass of the storage bodies is composed of this protein. A minor protein sequence related to the 17-kDa subunit of vacuolar H(+)-ATPase is also found in storage bodies isolated from pancreas. As in humans and cattle, the ovine protein is the product of two expressed genes named P1 and P2. In normal and diseased animals there are no differences in sequences between P1 cDNAs or P2 cDNAs, nor do levels of mRNAs in liver for P1 or P2 differ substantially between normal and diseased animals. Both normal and diseased sheep also express a spliced pseudogene encoding amino acids 1 to 31 of the mitochondrial import presequence. The peptides they encode differ by one amino acid; arginine-23 is changed to glutamine in the diseased sheep. Storage bodies isolated from brains and pancreas of children affected with the juvenile and late infantile forms of ceroid lipofuscinosis also contain large amounts of material that is identical to subunit c of ATP synthase. However, the protein is not present in storage bodies isolated from brains of patients affected with the infantile form of the disease, and these storage bodies contain other unidentified proteins. It is possible that the cause of ovine, juvenile and late infantile ceroid lipofuscinoses is related to a defect in degradation of the subunit c of mitochondrial ATP synthase.

Complications of rectal suction biopsy.

Complications following use of the rectal suction biopsy technique in the diagnosis of Hirschsprung's disease, are rare. In a series of 1,340 consecutive biopsies, complications included three clinical perforations of the bowel, one resulting in death, and three rectal hemorrhages requiring transfusion. A plea is made for the use of greater care in this technique.

Neuronal colonic dysplasia: an unusual association of Hirschsprung's disease.

A case of neuronal colonic dysplasia in association with Hirschsprung's disease is described. It is suggested that this condition be borne in mind when investigating operated Hirschsprung's cases having residual symptoms.