Physician/Scientist

Diagnosis and Treatment

Diagnosis

Clinical Diagnosis

Patients with Type A NPD present with hepatosplenomegaly, feeding problems, and failure to thrive in early infancy. The organomegaly is detectable in the first months of life. Neurologic involvement may be noted by 3 to 6 months of age. Developmental delay and/or loss of developmental milestones should signal the physician to consider NPD in the differential diagnosis. About 50 percent of the Type A infants have cherry-red macular spots, and this finding is often used to rule out infantile-onset Type 2 Gaucher disease. In Type B NPD patients, splenomegaly is usually the first manifestation detected. In most Type B NPD patients, the splenic enlargement is noted in early childhood; however, in very mild cases the enlargement may be subtle, and detection may be delayed into adolescence or adulthood.

The presence of the characteristic NPD cells in the bone marrow aspirates supports the diagnosis of Types A and B NPD. However, patients with Types C and D NPD also have extensive infiltration of NPD cells in the bone marrow, and thus, all suspect cases should be evaluated enzymatically to confirm the clinical diagnosis.

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Enzymatic Diagnosis

Types A and B NPD may be readily diagnosed by the markedly deficient activity of ASM in peripheral leukocytes, cultured fibroblasts, and/or lymphoblasts. In contrast, patients with Types C and D NPD will have somewhat decreased ASM activities, while patients with Gaucher disease and other storage disorders presenting with hepatosplenomegaly and/or neurologic involvement will have normal or near normal levels of ASM activity. Numerous radioactive, fluorescent, and colorimetric substrates have been used to measure ASM activity. In general, the in vitro residual ASM activities in isolated peripheral leukocytes or in cultured fibroblasts and/or lymphoblasts from Type A and B NPD patients range from less than 1 percent to a maximum of about 10 percent. Patients with Type B NPD may have slightly higher levels of residual ASM activity than those with Type A disease, but this phenomenon is not a reliable measure for predicting phenotypic severity. In addition to the in vitro assays, various cell loading (i.e., in situ) assays have been developed to determine ASM activity. In general, the levels of residual ASM activity determined with these in situ assays mimic those found with the in vitro systems.

Although the successful enzymatic identification of NPD carriers has been reported, many investigators have found that heterozygote detection by in vitro or in situ determination of the ASM activity is problematic. The major difficulty is the significant overlap between the low normal and high heterozygote ranges in peripheral leukocytes and in cultured cells.

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Molecular Diagnosis

As noted above, the R496L, L302P, and fsP330 mutations account for about 92 percent of the mutant alleles in Ashkenazic Jewish Type A NPD patients, facilitating the rapid identification of the molecular lesions in this population. PCR conditions have been developed to amplify the ASM genomic regions containing these mutations, and allele-specific oligonucleotides and/or diagnostic restriction enzyme tests are available. The R608 mutation has been identified in two of the four alleles analyzed from Ashkenazic Jewish Type B NPD patients. However, since the sample size is small, the true frequency of this mutation remains unknown, and additional Ashkenazic Jewish Type B NPD patients must be tested. In contrast, the non-Jewish Type A and B NPD patients have each had unique mutations, with the exception of R608.

In families in which the specific molecular lesion has been identified, family members can be accurately tested for heterozygote status by DNA analysis. Since the detectability of the Type A mutations is over 90 percent among the Ashkenazic Jewish patients, molecular carrier screening can be undertaken in this population.

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Prenatal Diagnosis

Prenatal diagnosis of NPD may be reliably made by the measurement of ASM activities in cultured amniocytes or chorionic villi. Measurement of sphingomyelin turnover by cultured amniocytes has been used to diagnose Type A NPD as well, but measurement of ASM residual activity is a more direct and reliable test. In families in which the specific ASM mutation has been identified, molecular analysis of the fetal cells can provide the specific diagnosis or serve as a confirmatory test. Analyses of tissues from affected fetuses terminated in the second trimester have shown five- to tenfold increases in the level of sphingomyelin in liver and spleen. Elevations of bis(monoacylglycero)phosphate also have been noted in the tissues of affected fetuses.

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