In 2001, Millat et al. studied the genotype/phenotype correlations in two patients from different Algerian families with NPC2. Fibroblast strains were used in the study and diagnosis of NPC had been assessed by filipin staining and by measurement of LDL- induced cholesteryl-ester formation. Molecular analysis revealed the presence of a nonsense homozygous mutation, E20X (glu20 to ter), in one patient and a splice mutation, IVS2+5G-A, in the other (a female alive at age 21 years). Homozygosity of the E20X mutation was associated with a severe rapid disease course; pronounced lung involvement leading to death from respiratory failure. On the other hand, the splice mutation was confirmed to be in a homozygous state by sequencing the genomic DNA of both consanguineous parents. The female patient with the IVS2+5G-A mutation was found to have splenomegaly at age 6.5 years, school problems, epileptic fits beginning at age 11 years, mental retardation, and slow progression, but was still able to walk normally at age 21 years. Remarkably an affected sister with a similar course was still alive at age 27 years. In two independent experiments, the electrophoretic profile of RT-PCR products from RNA isolated from the patient with splice mutation showed multiple bands. RT-PCR products therefore were subcloned, and 32 subclones were analyzed by sequencing. The analysis revealed 30 abnormally spliced cDNAs and two normally spliced cDNAs. Among the 30 mutant cDNAs, only three classes were observed- cDNA with a deletion of the last 76 bp of exon 2 and cDNA with a 10-bp insertion between exons 2 and 3.