Mutations in NPC1 highlight a conserved NPC1-specific cysteine-rich domain

Abstract

Niemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3' portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.

Figures

Figure 1

NPC1 mutations. Maps of (a) the 3′ end of the NPC1 gene and (b) the cDNA are shown. The positions of exons A–H are represented by squares, and introns are represented by lines. The positions of two intronic mutations are indicated. c, Diagram of the Npc1 protein aligned with the cDNA, showing the correspondence between the exons and their encoded amino acid sequence. Predicted domains within the Npc1 protein are indicated by squares shaded as follows: squares with diagonal stripes represent signal sequence; light gray–shaded squares, leucine zipper; dark gray–shaded squares, TM regions; and blackened squares, lysosomal targeting signal (Carstea et al. 1997). Regions of the Npc1 protein sharing >25% amino acid identity with the human Ptch1 protein are underlined. The positions of all reported NPC1 mutations in the coding sequence are represented along the protein as follows: blackened circles, missense mutations; unblackened triangles, deletions; and carats, insertions. Unless otherwise indicated, deletions and insertions result in frameshifts that introduce premature stop codons. d, Alignment of the amino acid sequence for the human (Hu) Npc1 domain between TM spans 9 and 10 with the corresponding regions from Npc1-related sequences from mouse (Mus Npc1), S. cerevisiae (Sc Ncr1), A. thaliana (GenBank accession number AL035539), and C. elegans (GenBank accession numbers U53340 and L11247). Asterisks indicate an identical residue at that position in all six sequences; dots, conservative amino acid substitutions; diamond, a position where a cysteine occurs in all sequences except that of A. thaliana, for which a histidine is substituted; dashes, gaps that are introduced to maximize the alignment; blackened circles above the alignment (with the letter, above the blackened circle, indicating the amino acid substitution), positions of missense mutations identified in this region in patients with Niemann-Pick type II disease; and unblackened triangles, positions of frameshifts created by deletions.

Figure 2

Comparison of human Npc1 and Ptch1 proteins. A model for the organization of Npc1 within the lysosomal membrane is contrasted with the proposed topology of human Ptch1 in the plasma membrane (Goodrich et al. ; Johnson et al. 1996). The model is a revision of that presented by Beachy et al. (1997). In the bottom portion of this figure, predicted TM domains for Npc1 (Carstea et al. 1997) and Ptch1 are numbered and are shown as cylinders. Stretches of amino acid sequence sharing >25% identity between the two proteins are gray shaded. A region with homology to sterol-sensing domains in Ptch1 (TM spans 2–6), HMG-CoA reductase, and SCAP (Carstea et al. 1997) is indicated. The cysteine-rich Npc1-specific domain is overlined and is shown in an expanded form at the top of the figure. To allow the conserved cysteine residues to be in close proximity to one another, the domain is depicted as three hypothetical loops. Blackened circles represent cysteine residues, and gray-shaded circles represent other positions conserved between human Npc1 and the five Npc1-related sequences. Conserved amino acid–sequence motifs in this domain are indicated by the single-letter code for the amino acid—by h for hydrophobic residues or by x for any amino acid.