Genomic DNA was isolated from cultured skin fibroblasts and screened for the 4 common acid β-glucosidase mutations (N370S, L444P, 84GG, and IVS2+1) by means of polymerase chain reaction (PCR) amplification and restriction digestion at natural or created sites.24- 28 The patient had 1 allele carrying the L444P mutation. The initial screening method could not differentiate between simple, singly mutated L444P alleles and several complex L444P-bearing alleles found in approximately 1.5% of Jewish and 3.5% of non-Jewish patients. To determine if the L444P mutation was present alone or as 1 of 3 known L444P-containing complex alleles,29 the complete acid β-glucosidase coding region and adjacent intron-exon boundaries were amplified by PCR and sequenced. Sequencing revealed a C→T base substitution at complementary DNA (cDNA) position 1448 (genomic position 7319) encoding the leucine→proline substitution at position 444 (L444P) and 4 additional nucleotide substitutions producing the following 3 missense mutations and a polymorphism: (1) a G→T transversion at cDNA position 1297 (genomic position 6799) encoding a valine→leucine substitution at amino acid 394 (V394L); (2) a G→A transition at cDNA position 1342 (genomic position 6844) encoding the substitution of an aspartate by hisitidine at residue 409 (D409H); (3) a G→C transversion at cDNA position 1483 (genomic position 7354) encoding the substitution of an alanine by proline at residue 456 (A456P); and (4) a polymorphic G→C transversion at cDNA position 1497 (genomic position 7368) in codon 460 (V460V). These results were consistent with earlier studies30 that, using dot blot analysis of genomic DNA from the patient and both parents, demonstrated that the V394L mutation came from the mother and that the 4 other mutations derived from the paternal allele. Thus, the mutation analysis identified 2 mutant alleles, one with a V394L mutation and the other with the lesion RecTL (D409H + L444P + A456P + V460V), which resulted from a recombination event with the pseudogene located 16 kilobases downstream from the structural gene.31 The V394L missense allele, as well as the 3 individual missense mutations (D409H, L444P, and A456P) of the complex RecTL allele, were expressed in the baculovirus system and characterized. The mutant proteins expressed from the D409H, L444P, and A456P alleles were severely compromised, with turnover rates of less than 0.2%, 5.0%, and greater than 0.03% of the healthy enzyme, respectively. In addition, immunoblotting studies (using polyclonal anti–acid β-glucosidase antibodies) of these 3 expressed mutant proteins consistently produced lighter signals than that of the expressed healthy enzyme demonstrating reduced stability. These results indicated that essentially no residual activity was derived from the multiply mutated RecTL allele. The V394L allele expressed a stable protein product with a catalytic efficiency that was approximately 8.5-fold lower than that of the healthy enzyme. In addition to the reduced turnover rate, the V394L mutant protein had been shown32 to have reduced stability at 0°C (half-life [t1/2] = 3.9 minutes; normal = 11 minutes) and pH of 3.5 (t1/2 = 13 minutes; normal = 30 minutes), suggesting that the intracellular lysosomal activity expressed from this allele would be very compromised. Thus, the combination of the inactive RecTL allele with the pH-sensitive V394L allele produced very little, but detectable, functional enzyme, consistent with the observed type 3 phenotype.