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.

Niemann-Pick type D (NPD) disease is a progressive neurodegenerative disorder characterized by the accumulation of tissue cholesterol and sphingomyelin. This disorder is relatively common in southwestern Nova Scotia, because of a founder effect. Our previous studies, using classic linkage analysis of this large extended kindred, defined the critical gene region to a 13-cM chromosome segment between D18S40 and D18S66. A recently isolated gene from this region, NPC1, is mutated in the majority of patients with Niemann-Pick type C disease. We have identified a point mutation within this gene (G3097-->T; Gly992-->Trp) that shows complete linkage disequilibrium with NPD, confirming that NPD is an allelic variant of NPC1.

We have isolated a clustered gene family in D. melanogaster that codes for trypsin-like enzymes. The gene family has been localized to 47D-F by in situ hybridization to polytene chromosomes. The four genes in the family are transcribed in alternating orientations, and code for 1000 nt mRNAs. Transcripts are present at all stages of the life cycle. In situ hybridization to mRNA in tissue sections of third instar larvae showed that transcripts were restricted to the mid-gut. One gene was sequenced. The translated amino acid sequence of the proposed active enzyme is 42% homologous to bovine trypsin. Regions of functional importance are more strongly conserved. These include the active site residues asp102, his57, ser195, and the residue asp189 which is reputed to bind the basic residue at the substrate cleavage site. The activation peptide is not homologous to that of most vertebrate trypsins, suggesting a modified activation mechanism. The sequence further strengthens the hypothesis that the chymotrypsin cleavage specificity developed separately in the vertebrates and invertebrates.

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Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH , genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%) , trisomy 12 (7%) , ATM deletion (6%) , 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes , the most common being 19p13 (LDLR and CDKN2D). Moreover , the cost for MLPA analysis, including technical time and reagents , is 86% less than FISH. In conclusion , cytogenetic abnormalities are a common finding in CLL patients , and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.