Norah R. McCabe

Rearrangement of the MLL Gene in Acute Lymphoblastic and Acute Myeloid Leukemias with 11q23 Chromosomal Translocations

BACKGROUND Translocations involving chromosome band 11q23 are very frequent in both acute lymphoblastic and acute myeloid leukemias and are the most common genetic alteration in infants with leukemia. In all age groups and all phenotypes of leukemia, an 11q23 translocation carries a poor prognosis. A major question has been whether one or several genes on band 11q23 are implicated in these leukemias. Previously, we identified the chromosomal breakpoint region in leukemias with the common 11q23 translocations and subsequently cloned a gene named MLL that spans the 11q23 breakpoint. METHODS We isolated a 0.74-kb BamHI fragment from a complementary DAN (cDNA) clone of the MLL gene. To determine the incidence of MLL rearrangements in patients with 11q23 abnormalities, we analyzed DNA from 61 patients with acute leukemia, 3 cell lines derived from such patients, and 20 patients with non-Hodgkins lymphoma and 11q23 aberrations. RESULTS The 0.74-kb cDNA probe detected DNA rearrangements in the MLL gene in 58 of the patients with leukemia, in the 3 cell lines, and in 3 of the patients with lymphoma. All the breaks occurred in an 8.3-kb breakpoint cluster region within the MLL gene. The probe identified DNA rearrangements in all 48 patients with the five common 11q23 translocations involving chromosomes 4, 6, 9, and 19, as well as in 16 patients with uncommon 11q23 aberrations. Twenty-one different chromosomal breakpoints involving the MLL gene were detected. CONCLUSIONS MLL gene rearrangements were detected with a single probe and a single restriction-enzyme digest in all DNA samples from patients with the common 11q23 translocations as well as in 16 patients or cell lines with other 11q23 anomalies. The ability to detect an MLL gene rearrangement rapidly and reliably, especially in patients with limited material for cytogenetic analysis, should make it possible to identify patients who have a poor prognosis and therefore require aggressive chemotherapy or marrow transplantation.

Molecular basis of an adult form of β-hexosaminidase B deficiency with motor neuron disease

A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta-Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons.

A study of the heterogenous structure of guinea pig lysosomal β-mannosidase using a polyclonal antibody

Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.

Preferential inhibition of lysosomal beta-mannosidase by sucrose

The lysosomal storage disease beta-mannosidosis, described in both goats and humans, can be detected by measuring a deficiency in hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannoside. An inhibitor of guinea pig beta-mannosidase (beta-man) activity was detected when tissue was homogenized in phosphate-buffered-saline (pH 7.4) containing 0.25 mol/l sucrose. The existence of such an inhibitor was apparent when the enzyme was immunoprecipitated from tissue using a specific beta-man polyclonal antibody. There was up to a threefold increase in activity in the immunoprecipitated enzyme (antibody-enzyme complex) compared to the activity of the nonimmunoprecipitated enzyme. An extensive study was therefore undertaken to determine the nature and specificity of this inhibitor by analyzing the effect of a range of metal ions and sugars on beta-man activity compared to other lysosomal hydrolase activities. Although ferrous, ferric, cobalt, and manganese ions were highly inhibitory to beta-man, they also inhibited other lysosomal hydrolases to a similar extent. Likewise, mannose inhibited both alpha- and beta-man activities equally. The only compound to specifically inhibit beta-man in a manner similar to that observed in the tissue homogenate was glucosyl(beta, 2)fructofuranoside (sucrose). This is an important finding in that tissue samples are commonly prepared in buffers containing sucrose and this could lead to a wrong diagnosis of beta-man deficiency. In order to determine if the absence of an activator factor or alternatively the presence of a specific inhibitor was a contributing factor in the lack of beta-man activity in cultured fibroblasts from affected humans and goats, mixing studies with normal and affected cell extracts were performed but no restoration or inhibition of beta-man activity was found.

Molecular heterogeneity in lysosomal storage diseases

The availability of specific antibodies and cDNA probes for lysosomal hydrolases has revealed unexpected heterogeneity among the human inherited lysosomal storage diseases. Using alpha-fucosidase and N-acetyl-beta-D-hexosaminidase deficiency variants as examples, it has been determined that a lysosomal hydrolase deficiency can result from DNA deletion mutations, failure to synthesize mRNA because of defective splicing, posttranslational defects in assembly, and synthesis of a precursor enzyme that is prematurely proteolytically degraded through lack of a protective protein. In some cases (fucosidosis), the different genotypes cannot be distinguished phenotypically, whereas in others (beta-hexosaminidoses) the phenotypes can range from infantile neurodegeneration through juvenile motor neuron disease to adult neurodysfunction. Biochemical studies on both diseases have revealed several distinct genotypes. We show that some forms of fucosidosis result from unstable enzyme that can be stabilized by protease inhibitors, whereas partial beta-hexosaminidase deficiencies cannot be corrected by these protease inhibitors.