Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

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The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in ''crew-cut'' bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.Several genetic determinants encoding different fimbrial structures have been identified in pathogenic strains of Escherichia coli. Expression of fimbriae (also denoted pili) on the bacteria is thought to play an essential role in the pathogenic process leading to infection by enabling the bacteria to adhere to epithelial surfaces. A common property of many of these determinants is the organization of the genes into polycistronic operons. These operons determine the production of one major structural protein (the pilin), some minor pilins, and other proteins involved in the assembly process of the fimbriae (37). The major structural proteins of the different determinants are expressed at levels markedly higher than those of the other proteins of the operons. Differential mRNA stability and mRNA processing have been suggested to be mechanisms to achieve the observed differential expression of genes in some E. coli pili determinants (3,7,17,24,28,39).pap gene clusters are commonly found in uropathogenic E. coli isolates. Studies of pap genes (papBAHCDJKEFG) from E. coli J96 suggest that expression includes mRNA processing, differential mRNA stability, and partial termination or attenuation (3, 28, 39). The two first cistrons expressed from the papB promoter, papB and papA, are cotranscribed to mRNA (mRNA-BA). A minor fraction of transcription proceeds past a terminator signal in...