Accurate diagnosis is imperative for genetic counseling for future pregnancies, because most of the LSDs are autosomal recessively inherited. There are many ways to diagnose LSDs (). With the development of new treatments for several of the LSDs, the diagnostic requirements are also changing. The efficacy of many of the proposed treatments relies heavily on early detection and initiation of treatment before the onset of irreversible pathology.137,138
Although many of the clinical presentations of different LSDs primarily result from substrate storage, these presentations vary greatly depending on the type, quantity, and site of the accumulated storage material. Because there is an overlap of clinical features in many of the LSDs, it is difficult to establish a diagnosis solely on the basis of clinical presentation. Fortunately, different accurate laboratory assays based on detection of the storage product, enzymatic assays, and DNA diagnostics have been developed. There are also biomarkers such as chitotriosidase that, although not optimally specific, can help monitor disease load. For example, in Gaucher disease, chitotriosidase levels decrease after ERT.
Urine screens that test for elevated levels of secreted substrate material are used routinely to examine the pattern of glycosaminoglycans and oligosaccharides in patients suspected of having MPS or disorders that present with oligosacchariduria, such as I-cell disease, mucolipidosis type 3, GM1 gangliosidosis, GM2 gangliosidosis type 2, fucosidosis, α
-mannosidosis, sialidosis, galactosialidosis, and ISSD. After determining that the level of glycosaminoglycans is elevated, electrophoresis can further support the diagnosis of the MPSs, although the definitive diagnosis is made by enzyme analysis of either leukocytes or cultured skin fibroblasts. Although urine screens are very sensitive, there have been reports of affected individuals with normal urine screens; thus, when there is a strong index of suspicion, normal urine screen results should still be followed by enzyme analysis.139–141
Generally, panels of enzyme activity assays are performed on a combination of leukocytes and plasma and predominantly include enzymes involved in the digestion of glycosphingolipids and oligosaccharides. Diseases tested for in these panels include Gaucher disease, Niemann-Pick disease types A and B, acid lipase deficiency, GM1 and GM2 gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucolipidosis type 2 and 3, fucosidosis, α
-mannosidosis, MPS type VII, and Schindler disease. Although measurement of enzyme activity in leukocytes and plasma enables the diagnosis of most LSDs in affected patients, a proportion may not be detected by this method. For example, in sialidosis and Pompe disease, the distinction between the normal and affected range in leukocytes can be very narrow, and the diagnostic analysis should be performed on other tissues or cultured fibroblasts. When leukocyte assays are not reliable, another method of enzyme analysis is to assay individual hair roots that develop from progenitor cells.138
A number of the LSDs result from deficiencies in secondary proteins or enzymes where the defect is in the activator protein. In these cases, the diagnosis can be achieved by examining the level of substrate secreted in the urine, or the rate of radiolabeled substrate turnover can be determined in cultured cells by substrate-loading tests. Ultimately, the relevant gene can be evaluated by mutation analysis. One of the examples for secondary protein deficiency is Niemann-Pick type C, which results from defective cholesterol transport in the lysosomal pathway and is diagnosed by analysis of cholesterol processing and accumulation in cultured fibroblasts.
Molecular analysis is rarely used as the primary screening tool for the diagnosis of LSDs. However, molecular analysis plays an important role with respect to carrier and prenatal testing for a variety of LSDs. Mutation data can also enable a rapid and accurate prenatal diagnosis. Because the demand for preimplantation diagnosis is rapidly increasing, it is very important that the causative LSD mutations be identified to enable the implantation of nonaffected fetuses. In a gene with several common mutations, this can be achieved more readily. In other disorders, however, the mutations identified may be novel or very rare. In cases when there is a strong diagnostic suspicion, sequencing of the relevant genes can be used to detect mutations. However, establishing that the nucleotide change identified is pathologic, rather than a mere polymorphism, can be challenging.138
Population screening for the LSDs is not performed routinely except for high-risk ethnic groups, for which screening for specific disorders may be appropriate, such as Tay-Sachs disease in the Ashkenazi Jewish population. Once a proband is identified in a given family, definitive carrier testing can be performed if the causative mutations are known.
Because of the early presentation of many of the LSDs, and the relative severity of these disorders, prenatal diagnosis is important for future pregnancies. Diseases that can be detected by enzymatic or biochemical changes in cultured cells can be detected using cultured chorionic villous cells or amniocytes for prenatal diagnosis. In many cases, direct analysis of chorionic villous tissue is also diagnostic, providing rapid and accurate results early in gestation. However, because some disorders can have pseudodeficiencies, it is often advisable to perform biochemical analysis on parental cultured cells, as well, to prevent a false-positive diagnosis.138
Whenever the causative gene is known, prenatal diagnosis can be made by mutation analysis. It is important that parental DNA is analyzed before prenatal testing to ensure that each parent carries 1 of the known causative mutations and that a corresponding normal allele can be detected also.
There are a few methods for the determination of enzymatic activities that have been developed recently on the basis of elution of the enzyme from a dried blood spot, followed by an assay of the enzyme activity by using fluorescent or radiolabeled substrates. Another alternative is the immune capture of the enzyme before the determination of enzyme activity. In addition, monoclonal and polyclonal antibodies can be used for the immune quantification of specific lysosomal proteins from biological samples, including dried blood spots. These assays provide a convenient and economical means for diagnosis and may have increasing importance as newborn screening for these disorders is considered.142,143
Electrospray ionization tandem mass spectrometry has been used effectively to investigate stored substrates in a number of the LSDs. This method has great potential and may enable the monitoring of responses to therapy.144,145
It is important to consider more than 1 type of assay as confirmation in any investigation of a patient suspected of having an LSD.
The decision regarding the advisability of newborn screening for the LSDs is complex. In addition to the requirements for a sensitive and specific assay, there are important ethical, economic, and counseling implications. Because the different LSDs have differing prognoses and therapeutic options, each disorder should be considered individually when deciding whether to implement newborn screening. Currently, screening for Krabbe and Pompe diseases are under discussion because of the therapeutic advances in these disorders, and it is likely that in the near future screening for other LSDs will be explored.