Niemann–Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative disorder characterised by intracellular accumulation of unesterified cholesterol and gangliosides within the endosomal–lysosomal system.¹ The incidence is estimated to be about 1 in 100 000 live births.^2,3 NPC is clinically heterogeneous and there is a broad spectrum of phenotypes with respect to the age of onset, organomegaly, and neurological disease. In most cases, neurological symptoms appear between the ages of 4 and 10 years,⁴ although the timing of onset can range from perinatal to adulthood. The neurological abnormalities develop insidiously and are manifested as ataxia, mental deterioration, psychomotor retardation, and seizures. Pyramidal tract signs, cerebellar ataxia, and extrapyramidal features such as dystonia are common. Supranuclear ophthalmoplegia with paralysis of down gaze is characteristic in patients with NPC. Cataplexy and various types of seizure may also occur during the evolution of the disease.⁵

Cultured fibroblasts from patients with NPC show a significant reduction in the rate of cholesterol esterification.⁶ Filipin staining of these cells reveals the abnormal accumulation of intracellular cholesterol, as evidenced by intense perinuclear punctate staining. In addition, cerebral neurones show the presence of neurofibrillary tangles, which are immunologically and ultrastructurally similar to those found in the brains of patients affected by Alzheimer’s disease.⁷ Some patients have cholestatic jaundice, and the infiltration of foamy histiocytes (Niemann–Pick cells) can be identified in the bone marrow.

NPC is genetically heterogeneous with two complementation groups.⁸ Mutations of the NPC1 gene have been identified in ∼95% of the patients with NPC.^9,10 The NPC1 gene, located on chromosome 18q11–12, spans more than 47 kb and contains 25 exons.¹¹ It encodes a 1278 amino acid protein with 13 transmembrane domains, three large luminal hydrophilic loops, six cytoplasmic loops, and a cytoplasmic tail with a dileucine motif.^12,13 The third luminal loop contains an NPC1 specific cysteine-rich domain (residues 855–1098). The NPC1 protein also contains a sterol sensing domain (residues 616–797), which is homologous to those found in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage activating protein.¹⁴ Disease causing mutations are spread throughout the NPC1 gene, although there is a concentration of mutations within the cysteine-rich domain, with an apparent hot spot for mutations between residues 927 and 958.¹⁵ In addition, the region between amino acids 1038 and 1253, which shares 35% identity with a portion of the Patched 1 (PTC1) protein, also contains a significant proportion of mutations.¹⁶ Most molecular defects of NPC1 involve only a few nucleotides such as small deletions, small insertions, or missense mutations (see the human mutation database¹⁷). The functional implications of the mutations to NPC1 are not yet clear; however, sterol homoeostasis dysfunction, especially the blockage of cholesterol transport between the lysosome and sterol regulatory machinery leading to the excessive accumulation of cholesterol, has been proposed to be the major cause of neuronal apoptosis in NPC.¹⁸

Based on an analysis of 134 cases, Vanier et al showed that diagnosis of NPC is best reached by the combined demonstration of a deficient induction of esterification and intravesicular cholesterol storage by filipin staining.¹⁹ However, identification of the molecular defect is important for confirming the diagnosis of NPC as well as enabling rapid and accurate prenatal diagnosis. Here, we describe the analysis of the NPC1 gene in five Taiwanese/Chinese patients with NPC, resulting in the detection of six novel NPC1 mutations.

PATIENTS AND METHODS

Patients

Five index patients and their family were studied. All families were of Taiwanese/Chinese origin. Ethical approval for genetic studies in Niemann–Pick disease type C (NPC) was obtained from the National Taiwan University Hospital Ethics Committee.

All index cases were diagnosed as having NPC based on clinical features, pathology in bone marrow, and biochemical analysis of cultured fibroblasts using filipin staining and cholesterol esterification. None of the families had a history of consanguinity. In all cases the disease started early in life, usually in the first decade (table 1). Hepatosplenomegaly was noted in all patients. The neurological features were highly variable among the families. However, patients within the same family had a similar neurological presentation (see table 1, patients 1, 4, and 5). The neurological manifestations included cerebellar ataxia, cerebral atrophy with psychomotor retardation, speech and mental function deterioration, and pyramidal as well as extrapyramidal signs (see table 1). Neuroimaging studies demonstrated dysgenesis of the corpus callosum with cerebellar or cerebral atrophy in patients 2 and 4. Non-specific high signals in T2-weighted images were seen in the white matter of brain and spinal cord of patients 1 and 5, respectively (fig 1C). Bone marrow biopsies of all index patients showed histiocyte infiltration (fig 1A). Filipin staining of skin fibroblasts and cholesterol esterification assays were performed in the National Referral Laboratory, Adelaide, Australia (Dr M J Fietz). All patients had accumulation of intracellular cholesterol similar to that shown in fig 1B. The rate of cholesterol esterification was also significantly reduced in these patients (table 1, normal range >3 pmol/hour/mg protein).

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Figure 1

 Pathological and neuroimaging manifestations in patients with NPC. (A) Light micrograph of a bone marrow aspirate showing the presence of a foamy histiocyte. (B) Presence of perinuclear non-esterified cholesterol deposits in cultured fibroblasts visualised by fluorescence microscopy after filipin staining. (C) Axial T2-weighted magnetic resonance image showing non-specific hyperintense areas in the white matter.

METHODS

DNA was extracted from leucocytes using a standard method. All the coding exons (26 amplicons) and the flanking regions of the NPC1 gene were amplified by polymerase chain reaction (PCR). The primer sequences are available on request. Heteroduplexes were screened by denaturing high performance liquid chromatography (DHPLC) using a Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc, San Jose, CA). The protocol for this has been described elsewhere.²⁰ Equal amounts of PCR product from the patient and a normal control were mixed and then denatured and renatured by cooling from 95 °C to 25 °C over 70 minutes. Exons showing the presence of a heteroduplex were sequenced using the BigDye Terminator sequencing chemistry (ABI) and the ABI3100 automatic DNA sequencer (Applied Biosystems, Foster City, CA).

Both filipin staining and cholesterol esterification assays were performed using modified versions of the methods of Vanier et al.¹⁵

RESULTS

All 26 exons of the NPC1 gene were amplified from each of the five index patients and subjected to DHPLC. This revealed the presence of heteroduplexes in all five patients. Sequencing demonstrated a total of six different mutations, 2213C>A (S738Stop), 2903A>G (N968S), 3044G>T (G1015V), 3100G>A (G1034R), 3761G>T (V1212L), and 1905del T/insATG (I635fs) (see table 1). The missense mutations were located in exons 19 (N968S), 21 (G1015V, G1034R), and 24 (V1212L). The nonsense mutation (S738Stop) resulted in a premature stop codon at position 738. Likewise, the frameshift mutation (I635fs) encoded a truncated protein with a premature stop codon at position 638. One of the missense mutations (V1212L) occurred in two index patients (patients 1 and 4). Patient 3 was homozygous for S738Stop while the other four mutations each accounted for a single allele in the cohort. Only one mutant allele each was identified in patients 2 and 4, G1034R and V1212L, respectively. The six mutations were absent in 100 Taiwanese controls. In addition to the mutations, six polymorphisms (H215R, I642M, I858V, N931N, R1266Q, and R1314R) were also identified.

DISCUSSION

As is seen in many lipid storage disorders, the clinical features of NPC are highly variable. There is considerable variation in the age at which symptoms first appear as well as in the progression and neurological manifestations of the disease. Generally, the later the neurological symptoms appear, the slower the progression of the disease. In our patients, the younger the age of onset, the more severe was the phenotype.

To date, more than 130 disease causing mutations have been reported for NPC and most of these are missense mutations (71%).¹⁵ Only a small number of frequently occurring mutations have been described, namely, I1061T and P1007A (in individuals of western European descent), R518Q (Japanese), and P474L (Italian). None of these was detected in the present study. More than a third of the NPC1 mutations are concentrated within the cysteine-rich luminal loop of NPC1. The cysteine-rich loop contains a ring-finger motif and is a likely site for functionally significant protein–protein interactions.^22,23 Three of the missense mutations detected in this study are located within the cysteine-rich loop (N968S, G1015V, and G1034R). However, none occurs in the hot spot region (residues 927–958). The fourth missense mutation, V1212L, occurs in transmembrane domain XII (TM XII), which is located in the mutation-rich region homologous to PTC1. Mutations I635fs and S738Stop introduce premature stop codons at amino acids 638 (TM IV) and 738 (TM VI), respectively. None of the six novel mutations was detected in 100 Taiwanese controls indicating that it is likely that these six mutant alleles are pathogenic.

For two of the five patients examined in this study only one mutation to the NPC1 gene was detected. Given the clinical and biochemical presentations of these patients, it is highly unlikely that both are true carriers of NPC. Therefore, it remains possible that a second point mutation in these patients was beyond the resolution of DHPLC and sequencing analysis. Alternatively, these mutations could have resulted from a large deletion or other genomic changes affecting either the amplification of the relevant exons or the overall structure of the gene. Amplification of the NPC1 cDNA or genomic Southern analysis could be used to examine these options further.

NPC is a complex neurological disorder with a broad range of clinical presentations, routinely diagnosed using biochemical analysis. Mutation screening provides an alternative means of confirming the diagnosis of NPC. In addition, the detection of both causative mutations allows carrier screening of family members as well as enabling rapid and accurate prenatal testing by mutation analysis of DNA from fetal cells.

In summary, we have described the mutation screening of five Taiwanese/Chinese patients with NPC, resulting in the detection of six novel NPC1 mutations. This is the first report of NPC1 mutations in patients from this ethnic background.