Chapter 153

## Abstract

Abstract

1. The GM2 gangliosidoses are a group of inherited disorders caused by excessive accumulation of ganglioside GM2 and related glycolipids in the lysosomes, mainly of neuronal cells. The enzymatic hydrolysis of ganglioside GM2 requires that it be complexed with a substrate-specific cofactor, the GM2 activator. There are two isoenzymes of β-hexosaminidase, Hex A, structure αβ, and Hex B, structure ββ where only Hex A can act on the ganglioside GM2/GM2 activator complex. Defects in any of three genes may lead to GM2 gangliosidosis: HEXA , which encodes the α-subunit of Hex A; HEXB , which encodes the β-subunit of Hex A and Hex B; or GM2A , which encodes the monomeric GM2 activator. There are three forms of GM2 gangliosidosis: (a) Tay-Sachs disease and variants, resulting from mutations of the HEXA gene, are associated with deficient activity of Hex A but normal Hex B; (b) Sandhoff disease and variants, resulting from mutations of the HEXB gene, are associated with deficient activity of both Hex A and Hex B; and (c) GM2 activator deficiency, due to mutation of the GM2A gene, is characterized by normal Hex A and Hex B but the inability to form a functional ganglioside GM2/GM2 activator complex. There are also pseudodeficient or clinically benign “disorders” characterized by biochemical defects of Hex A but functional activity toward ganglioside GM2. The gross pathology is very similar in Tay-Sachs disease, Sandhoff disease and GM2 activator deficiency, except that there is also involvement of the visceral organs in Sandhoff disease. The most pronounced cellular change is the presence of swollen neurons with storage material in lysosomes throughout the nervous system. Characteristic inclusions are the so-called membranous cytoplasmic bodies.

2. The hexosaminidases and GM2 activator are glycoproteins that are synthesized in the lumen of the endoplasmic reticulum (ER) and processed through the Golgi. They are transported to the lysosome via the mannose-6-phosphate receptor where they are processed further to their final “mature” forms. β-hexosaminidase A (Hex A) and β-hexosaminidase B (Hex B) hydrolyze a broad spectrum of substrates that are specific for terminal GlcNAc or GalNAc residues in β-linkage. Active site residues and the mechanism of catalysis are being revealed through mutagenesis, photoaffinity labeling with substrate analogs, and studies of chimeric α-β enzyme. In addition to the hydrolysis of ganglioside GM2 by only Hex A, both isoenzymes will catabolize glycoproteins, glycosaminoglycans, and glycolipids. They will also hydrolyze synthetic substrates of which the most sensitive and commonly used is β-GlcNAc derivative of 4-methylumbelliferone (4MUG). It is recognized by both Hex A and Hex B and does not require the GM2 activator. Using 4MUG, Hex A and Hex B can be distinguished by taking advantage of different thermal or other characteristics of the isoenzymes. A related compound cleaved by Hex A (and Hex S), but not Hex B, is 4-methylumbelliferyl-GlcNAc-6-sulfate (4MUGS). Substrate such as 4MUG or 4MUGS are used for routine diagnostic testing and for screening of carriers of the gene encoding the α-subunit of Hex A isoenzyme in humans (HEXA) or the gene encoding the β-subunit of Hex A and Hex B isoenzymes in humans (HEXB) alleles.

3. The clinical phenotypes associated with each of the different biochemical variants vary widely from infantile-onset, rapidly progressive neurodegenerative disease culminating in death before age 4 years (classical Tay-Sachs disease and Sandhoff disease, as well as GM2 activator deficiency) to later-onset, subacute or chronic, progressive neurologic conditions compatible with survival into childhood or teens (subacute form) or long survival (chronic form). Chronic forms include several different clinical phenotypes in which symptoms referable to one or another part of the central nervous system dominate, including progressive dystonia, spinocerebellar degeneration, motor neuron disease, or psychosis.

4. At least 92 specific HEXA , 26 HEXB , and 4 GM2A mutations have been characterized to date. The majority of mutations cause the severe, infantile-onset disease. Mutations causing subacute or chronic forms show correlation between the level of residual activity toward GM2 ganglioside and a combination of the level of ganglioside GM2 normally present in the cells in question. Several mutations are responsible for special subtypes of Hex A deficiency. The B1 subtype includes HEXA mutations in the putative catalytic domain of the α-subunit. They do not interfere with the synthesis of the Hex A heterodimer and leave intact Hex A activity toward 4MUG due to the functional β-subunit, but are inactive toward 4MUGS or GM2 ganglioside. Other mutations, causing the chronic disease phenotype, result in an unstable protein that may fail to associate with the β-subunit or are otherwise incompletely processed. Hex A pseudodeficiency is caused by point mutations that leave the Hex A with reduced but variable activity with synthetic substrates, but with sufficient ganglioside GM2 hydrolyzing activity to escape disease.

5. Heterozygotes for any one of the defects are asymptomatic; thus all GM2 gangliosidosis variants are inherited in an autosomal recessive fashion ( HEXA maps to chromosome 15 and HEXB and GM2A to chromosome 5). The availability of rapid and inexpensive methods for the identification of heterozygotes for Hex A deficiency has made possible large programs for family and population screening. Furthermore, when coupled with DNA-based diagnostics, mutations leading to infantile, subacute, or chronic disease can be distinguished. This has importance in recognizing the presence of pseudodeficiency alleles that are not easily distinguished biochemically. In the general population, heterozygote frequencies are estimated at 0.006 for HEXA mutations and 0.0036 for HEXB mutations. About 35 percent of alleles are accounted for by two mutations associated with the infantile phenotype and a further 5 percent by a prevalent chronic mutation. Of particular importance is that 35 percent of non-Jewish individuals are heterozygous for one of the two pseudodeficiency mutations. In some ethnic groups, much higher carrier frequencies are observed. Among the Ashkenazi Jewish people in North America and in Israel, the heterozygote frequency for HEXA mutations is 0.033. This includes two “infantile” mutations that account for 90 to 95 percent of the total, the chronic mutation that accounts for 3 percent, and the pseudodeficiency mutations that account for 2 percent of the alleles. Extensive genetic counseling and monitoring of fetuses at risk has reduced the incidence of Tay-Sachs disease in the Ashkenazi Jewish population by almost 90 percent.

6. Murine models for the three GM2 gangliosidoses have been generated by targeted gene disruption. Sandhoff mice, lacking Hex A and Hex B, accumulate ganglioside GM2 and glycolipid GA2 in nervous tissue, show a characteristic neuropathology and a progressive, severe neurodegenerative disease culminating in death at 4 to 4.5 months of age. Tay-Sachs mice, lacking only Hex A, accumulate GM2 to a lesser extent with negligible GA2, have a less extensive and more restricted neuropathology, and escape disease to at least 1 year of age. These mice bypass the Hex A defect by catabolizing accumulating GM2 via GA2 and lactosyl-ceramide by the action of sialidase and Hex B. The mouse model of GM2 activator deficiency results in a functional deficiency of Hex A and Hex B activities. These mice accumulate GM2 and a low amount of GA2 and have a motor deficit, but with a normal life span. The double-mutant mice, derived by crossbreeding, have deficient α and β subunits of Hex A. They lack Hex A, Hex B, and the Hex S α dimer characteristic of Sandhoff disease. In addition to their ganglioside storage disease, these mice show features of severe mucopolysaccharidosis and accumulate and excrete glycosaminoglycans, findings that underscore the importance of the Hex S residual activity in Sandhoff disease.

7. Specific therapy for GM2 gangliosidosis is not available to date, although all Hex A-deficiency variants can be diagnosed prenatally from amniotic fluid, cultured amniotic fluid cells, or chorionic villus biopsies. Several approaches to therapy are being investigated in animal or cell-culture models of the GM2 gangliosidoses. These approaches include enzyme replacement therapy, bone marrow and neural progenitor cell transplantation, gene therapy, and substrate deprivation therapy, the last by drug-mediated restriction of ganglioside biosynthesis in mutant mice.

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