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Niemann-Pick type C (NP-C) disease exhibits marked heterogeneity in its phenotype. This can pose diagnostic dilemmas and even delayed recognition of this condition.
To highlight the phenotypic variations and distinctive pathological and biochemical findings in this disorder.
Descriptive case studies.
Tertiary care children’s hospital and clinic.
Three cases of NP-C disease where diagnosis was delayed.
In each of the three cases the clinical presentation was varied, one as neonatal hepatitis, the second with megaloblastic anemia, chronic hepatitis and short stature, and the third with neonatal hepatitis and chronic respiratory failure. Definitive diagnosis was established in each case by demonstration of defective cholesterol esterification in skin fibroblasts.
In the clinical setting of neonatal hepatitis, hepatosplenomegaly and undiagnosed neurological symptoms, NP-C disease should be considered in the differential diagnosis. Electron microscopic examination of skin biopsy is an effective screening test, although the definitive diagnosis should be made by the cholesterol esterification assay and filipin staining.
La maladie de Niemann-Pick de type C (NP-C) arbore une hétérogénéité phénotypique, laquelle peut poser des dilemmes diagnostiques et même donner lieu à un diagnostic tardif.
Souligner les variations phénotypiques et les observations pathologiques et biochimiques propres à cette maladie.
Étude descriptive des cas.
Hôpitaux et cliniques de soins tertiaires pour enfants.
Trois cas de maladie de NP-C au diagnostic tardif.
Dans chacun des trois cas, la présentation clinique variait. Dans le premier cas, on observait une hépatite néonatale, dans le deuxième, une anémie mégaloblastique, une hépatite chronique et une petite taille et dans le troisième, une hépatite néonatale et une défaillance respiratoire chronique. On a posé un diagnostic définitif dans les trois cas en démontrant une estérification défectueuse du cholestérol sur des fibroblastes cutanés.
Le diagnostic différentiel de maladie de NP-C devrait être envisagé en milieu clinique en cas d’hépatite néonatale, d’hépatosplénomégalie et de symptômes neurologiques non diagnostiqués. Un examen au microscope électronique de la biopsie cutanée représente un test de dépistage efficace, bien que le diagnostic définitif doive reposer sur l’évaluation de l’estérification du cholestérol et sur une coloration à la filipine.
Inborn errors of metabolism, a group of rare disorders, are usually not considered as an initial diagnostic possibility in a child with multisystem involvement. In most instances, family physicians and paediatricians are the first to encounter such cases. Delay in recognition and failure to establish a diagnosis early can profoundly affect the implications for treatment and genetic counselling.
Niemann-Pick type C (NP-C) disease typifies such a clinical situation. It shows tremendous variability in presentation, both in terms of the age of onset as well as in the phenotype. Therefore, it is often possible for the diagnosis to be missed completely or delayed as illustrated by the three cases presented in this paper. We highlight the common and uncommon presentations of this metabolic disorder. Early diagnosis is vital because therapeutic options are limited for this disorder.
A boy, aged two years and nine months, was referred for evaluation of developmental delay and hepatosplenomegaly. He was born at full term to a 26-year-old Gravida 1 Para 0 woman and weighed 2580 g. He was of Iranian descent, and his parents were first cousins. Neonatal history was significant for jaundice. At five months, he developed asymptomatic pancytopenia without bruising, infection or bleeding. Massive hepatosplenomegaly was noted by one year of age. There was a delay in acquiring all milestones and a recent loss of developmental skills. He was able to walk with support, while language development was limited to single words. His general activity level was poor. Other medical problems included recurrent upper respiratory tract infections and lung collapse on several occasions. There were no bowel or urinary complaints.
Physical examination revealed a pleasant young boy with a weight of 13.5 kg (10th to 25th centile), height of 91 cm (10th to 25th centile) and head circumference of 49.5 cm (50th centile). He was nondysmorphic. There was no jaundice or anemia. He had a protruding abdomen and wasted extremities. There was significant hepatosplenomegaly. The liver was 9 cm below the costal margin, and the spleen was palpable in the right iliac fossa. There was no evidence of portal hypertension. Neurological examination revealed the child to be conscious and alert. He drooled continuously. There was no cherry red spot on fundus examination. Extraocular movements were normal in both horizontal and vertical gaze. He had generalized hypotonia, deep tendon reflexes were barely elicited, and plantar responses were flexor.
Initial evaluations were focused on the hepatosplenomegaly to elucidate a cause. Liver biopsy and bone marrow examinations were inconclusive. A sweat test was negative, and alpha-1 antitrypsin levels were normal. White blood cell sphingomyelinase levels were normal, mucopolysaccharides and oligosaccharides were undetected in the urine.
At this stage, both Gaucher’s disease and NP-C were considered in the differential diagnosis. The history of neonatal hepatitis suggested a strong likelihood of NP-C. A skin biopsy was done, and fibroblast (low density lipoprotein [LDL]) cholesterol esterification was found to be markedly defective at 4±4 pmoles cholesterol/mg protein (normal values for controls 3432±1729 pmoles cholesterol/mg protein) (1). Filipin staining was also positive, with storage of unesterified cholesterol in lysosomes of these cells, consistent with a diagnosis of NP-C.
His neurological condition has continued to deteriorate since diagnosis. By five years of age, he has shown a significant decline in motor skills and is bed-bound. He has gradually lost all comprehension of spoken language and is presently mute. In addition, he has developed seizures in the form of stiffening episodes. Electroencephalogram was abnormal with electrodecremental responses observed during seizures. Recent neurological examination confirmed the presence of marked spasticity and exaggerated deep tendon reflexes with extensor plantar responses. Prominent contractures have now developed at the ankles, knees and hips. A feeding gastrostomy tube has been placed, and he receives palliative care.
A 14-year-old boy presented with megaloblastic anemia of six to seven years’ duration, hepatosplenomegaly for three to four years’ duration, and short stature. He was relatively asymptomatic, with the exception of mild icterus and episodic epistaxis. Family history was not contributory. There was no history of consanguinity. Weight and height were below fifth centile. He had a grade 2/6 ejection systolic murmur, a palpable liver 3 to 4 cm below the right costal margin and a splenomegaly of 5 cm below left costal margin. Mitral regurgitation was demonstrable on echocardiography. His neurological examination was entirely normal.
Numerous investigations initially included normal bone marrow and a normal alpha-1 antitrypsin levels. Laboratory work-up for Wilson’s disease and viral hepatitis was unhelpful. Leukocyte sphingomyelinase and acid lipase were normal. A liver biopsy showed nonspecific findings with fatty change. A presumptive diagnosis of chronic active hepatitis was made. The hepatosplenomegaly persisted. During follow-up, he developed chronic thrombocytopenia, for which a splenectomy was performed. At surgery, a liver and a muscle biopsy were also obtained. The spleen showed lipid and lamellar aggregates on electron microscopy. The muscle showed excess lipid. On light microscopy, liver pathology was suggestive of chronic active hepatitis with Mallory bodies, fatty change and bridging fibrosis. Electron microscopy, however, showed storage of complex lipid in the form of membrane-bound whorled and lamellated aggregates and cholesterol crystals in the liver cell cytosol (Figure 1). Based on these results, NP-C disease was considered as a diagnostic possibility. Cultured skin fibroblasts analyzed for cholesterol esterification revealed values of 10.7±1.9 nmoles/24h/mg protein (control value of 29.7±8.1 nmoles/24h/mg protein) (2). Cholesterol esterification values are considered intermediate between severely affected and normal. He has completed grade 11 at age 24 years. No further details of his follow-up are available, other than he is malnourished (weight below the fifth centile) and exhibits a poor overall level of activity.
A female infant was born at 38 weeks gestation to a 26-year-old primigravida. She was noted to have abdominal distention after her first feeding. She developed splenomegaly and jaundice on second day of life. She was investigated and treated for presumed sepsis (cultures were negative). She was discharged home, only to be readmitted for splenomegaly and progressive jaundice. Two liver biopsies, one done at 23 days and one completed two weeks later, were consistent with neonatal hepatitis, progressing to cirrhosis. Electron microscopic studies were not carried out on the tissue. She was treated with prednisone and phenobarbital, and discharged at two months of age. At four months of age, attempts to taper prednisone led to respiratory failure requiring aggressive ventilation. She remained intubated and ventilated until age two years and three months. A lung biopsy showed alveolar exudate suggestive of pneumocystis infection, but no organism was identified.
During follow-up, her splenomegaly persisted, while the elevated bilirubin and hepatic enzymes normalized. Her developmental delay was thought to be related to her systemic illness. She was discharged at age two years and six months. At the time of discharge she did not require ventilatory assistance, but needed supplemental oxygen through a tracheostomy. She acquired the ability to speak simple sentences and could crawl. She was aware of her surroundings and interacted well. Over the next one-and-a-half years she had multiple hospitalizations due to respiratory complications. She continued to produce copious amounts of thick respiratory secretions. She developed progressive generalized weakness and lost her ability to speak. She was readmitted at age four years and six months for management of progressive pulmonary insufficiency. Chest x-ray suggested an interstitial lung disease. She required intermittent ventilation and ultimately did well with night-time ventilation only. A diagnosis of pulmonary alveolar proteinosis was entertained, but analysis of bronchoalveolar lavage fluid ruled out this diagnosis.
Hepatic and splenic enlargement did not regress. She was discharged after three months of hospital stay. During the next three years she had slow deterioration in muscle strength. She then developed epileptic seizures presenting as ‘drop attacks’. At the age of seven years and seven months, she became thrombocytopenic secondary to her hypersplenism. Sphingomyelinase activity was normal. A bone marrow aspirate showed foam cells. A muscle, sural nerve and a skin biopsy showed lamellar inclusions in various cell types consistent with NP-C (Figure 2). This was further confirmed by defective cholesterol esterification in cultured fibroblasts obtained from skin biopsy: 4.00±1.53 nmoles/24h/mg protein (normal value of 23.5±4.69 nmoles/24h/mg protein) (2). Neurological examination continues to show generalized muscle weakness and findings consistent with spastic diplegia. She requires both night-time and partial daytime ventilatory support.
NP-C, an autosomal recessive inborn error of metabolism, exhibits marked heterogeneity in phenotype and is characterized by a unique abnormality in cholesterol metabolism that differentiates it from the sphingomyelinase deficient forms of Niemann-Pick diseases A and B (3). Clinical presentations are listed in Table 1. NP-C presents in childhood with manifestations, such as hypotonia, failure to thrive and neonatal jaundice, that are frequently shared by many systemic disorders in childhood. In addition, less frequent symptoms of neurological (dystonia, supranuclear gaze palsy) or non-neurological involvement (nodular splenomegaly, pulmonary symptoms) occur. The diagnosis of NP-C disease, thus, rests on a high index of clinical suspicion and documentation of biochemical and histological abnormalities. Screening tests such as ultrastructural examination of skin can be extremely helpful. Loosely arranged lamellar structures admixed with electronlucent cytosomes found in the macrophages, pericytes, Schwann cells, smooth muscle cells and fibroblasts are highly suggestive of a cholesterol ester storage disorder. Skin biopsy findings are particularly of value if access to the specialized laboratories is unavailable for performing cholesterol esterification studies (4). Definitive laboratory testing includes fibroblast LDL cholesterol esterification assay which is defective (5). Filipin staining of the skin fibroblasts is also helpful. Filipin is a fluorescent probe that forms specific complexes with unesterified cholesterol (6). Complementation studies have led to identification of two groups of NP-C disease; a major (NP-C1) and a minor (NP-C2) group (7). Both forms of NP-C result from defective intracellular transport of cholesterol.
In the first case described, despite the classical presentation of neonatal hepatitis, developmental delay and hepatosplenomegaly, the diagnosis of NP-C was not a prominent consideration at the outset. Gaucher’s disease was considered in the differential diagnosis. However, on re-evaluation, history of neonatal hepatitis was thought to be more in favour of NP-C. Results from liver biopsy and bone marrow examination can be inconclusive, as with our patient. Foamy histiocytes may be seen and suggest the possibility of a storage disease, but their presence is not helpful in defining a precise cause. It was only through the defective LDL cholesterol esterification assay in the fibroblasts that the diagnosis in the first case was conclusively established. In a study by Kelly et al (8), 34 children (65%) of 52 children with NP-C had persistent cholestatic liver disease. Thus, unexplained neonatal hepatitis should prompt further investigations to rule out NP-C. Frequently, the jaundice subsides and the neurological features, such as cognitive decline, ataxia, dystonia and vertical supranuclear gaze palsy, become evident.
In the second case, the patient presented with hepatosplenomegaly and growth retardation, a common paediatric problem requiring an extensive work-up. This case was unusual for lack of neurological symptoms and findings. The clinical course was complicated by megaloblastic anemia. Initial liver biopsy suggested findings of chronic hepatitis; however, electron microscopic studies pointed towards a cholesterol ester storage disorder. Examination of splenic tissue also disclosed similar findings. Intermediate low values obtained on cholesterol esterification assay in comparison with the other two cases may account for a milder metabolic defect and, therefore, a lack of neurological features in adolescence. A filipin staining of the skin fibroblasts would have been of added benefit, but was not carried out.
Cholesterol esterification data differ significantly among our cases due to differences in the methodology used by the reference laboratories where the assays were carried out. However, it must be pointed out that biochemical heterogeneity in the degree of cholesterol esterification of NP-C has been confirmed in other studies (9). The findings suggest a correlation between milder esterification block and disease phenotype. In a larger study on kinetics of LDL-stimulated cholesterol ester formation and intravesicular accumulation of LDL-derived unesterified cholesterol through filipin staining on 125 patients with NP-C, three groups were identified (10). The largest group (86%), demonstrating the classic NP-C phenotype, showed profound alteration with esterification rates less than 10% of normal and numerous intensely fluorescent filipin granules. An intermediate group (7%) showed higher rates of cholesterol ester formation with more difficult to diagnose features, and a third ‘variant’ group (7%) presented with only slight alteration in esterification restricted to the early stages of LDL uptake, which was indistinguishable from heterozygotes. The third group showed abnormal cytochemical distribution of LDL-derived cholesterol (10,11). These findings and the different rates of cholesterol esterification taken together further confirm the biochemical heterogeneity of NP-C.
In the third case, the clinical course was complicated predominantly by pulmonary involvement. This has been described in Niemann-Pick A and B as well as NP-C2 type, although it is an infrequent presentation of NP-C1 (classic) (12). Initial findings suggestive of pulmonary alveolar proteinosis have been reported earlier by Kovesi et al (13) in their patient.
The therapeutic options for this disease are limited because neither drug therapy nor tissue transplantation has been found to reverse the disease process (14). Most patients do not survive beyond the second decade. Drug therapy to lower liver cholesterol has been tried (15). In this study, although the hepatic and plasma cholesterol were lowered by using different combinations of cholestyramine, lovastatin, nicotinic acid and dimethyl sulfoxide, there was no significant improvement in the neurological status of the patients (15). Supportive care of patients involves anticholinergic medications for dystonia, protryptyline hydrochloride for cataplexy and antiepileptic medications for seizures. Gastrostomy for feeding purposes, and speech and swallowing therapy are essential to avoid recurrent lung complications of pneumonia and collapse. Psychosocial support for the patient and family is paramount (16). Genetic counselling is an integral part of the management. Prenatal diagnosis is available; however, the results can be inconclusive and ambiguous if the defect is mild (17). Currently, cholesterol esterification is employed along with filipin staining on both amniotic fluid and chorionic villus samples.
The gene for the major group (NP-C1) has been recently cloned; the gene locus is on chromosome 18q11–q12 (18). NP-C1 appears to play a critical role in regulation of intracellular cholesterol trafficking. Further insight to this NP-C1 protein’s function has revealed homology with morphogen receptor PATCHED in Drosophila, a sterol, regulatory element binding protein and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the regulatory enzyme of de novo cholesterol biosynthesis, suggesting that it may be a receptor for proteins involved in cholesterol transport. Gene therapy remains a distant goal.
Awareness of this disorder among clinicians is the first step towards establishing an early diagnosis. The existence of biochemical heterogeneity as exemplified by our cases and others may account for the phenotypic heterogeneity seen in our cases, and the great variability in clinical features listed in Table 1. With the availability of definitive diagnostic testing and a 25% recurrence risk, NP-C disease, a neurometabolic condition, should be considered in the differential diagnosis when the clinical picture remains unexplained in multisystemic disorders, particularly in the presence of hepatosplenomegaly and neurological deterioration.
We thank Lisa Lee and Howard Gladney from the Electron Microscopy Laboratory, Memorial University, St John’s, Newfoundland for their technical help. We are grateful to Dr John W Callahan, Director of Lysosomal Diseases Laboratory, The Hospital for Sick Children, Toronto, Ontario and Dr PG Pentachev, Developmental Metabolic Neurology branch of NINDS(NIH), Bethesada, Maryland for the cholesterol esterification studies.