Status of Calpain In gene deleted carriers of Duchenne Muscular Dystrophy (DMD).

Though DNA testing gives precise diagnosis, molecular heterogeneity of disease makes testing an elaborate effort in Duchenne Muscular Dystrophy (DMD) in spite of the fact that 60°% of the cases are caused by deletions. The molecular diagnosis of carriers is elaborate needing quantitation of amplifed exonic products. Use of polymorphic markers for other mutations are also involved. We had reported in a large pedigree of DMD with one exonic deletion, the status of calpain by ELISA, CPK and deletion assessed by quantitative PCR (QPCR) in mothers and female siblings. The results pointed to the fact that calpain test is positive in true carriers (showing gene deletion). Status of catpain was assessed in many unrelated female members of DMD families carrying other exonic deletions, which was positive to calpain changes in true carriers.

Deletion analysis & calpain status for carrier detection in a family with Duchenne muscular dystrophy.

Eight females with a family history of Duchenne muscular dystrophy (DMD) were analysed for their carrier status by m-calpain test, which monitors the m-calpain (milli-calpain), a proteolytic enzyme in the platelets, using an ELISA technique. Four of the eight females were identified as carriers by virtue of their elevated enzyme levels as compared to control. DNA samples of these members were analysed to ascertain the carrier status, by PCR followed by dosage analysis by densitometry. DNA analysis confirmed the findings by calpain test, which underlines the reliability of this phenotypic test for carrier detection in DMD. Calpain test has been informative in a large group of patients and carriers tested so far. Since the calpain test is cost and labour effective, it is suited for routine and widespread screening purposes.

Analysis of sickle cell gene using polymerase chain reaction & restriction enzyme Bsu 361.

A 772bp DNA fragment from human beta-globin gene has been amplified by polymerase chain reaction (PCR) and subjected to restriction enzyme analysis using Bsu 361, an isoschizomer of restriction enzyme Mst II. This protocol has been designed basically to enhance the analytical facility for the detection of sickle cell mutation. A 430bp DNA fragment was found to be associated with the mutant locus, whereas 228bp and 202bp DNA fragments were generated from the normal locus. This difference of about 202bp in the resulting fragments from the mutant and normal loci has improved discriminatory power in the genotype analysis of the sickle cell mutation.

Calcium activated neutral protease & calcium ATPase in glucose-6-phosphate dehydrogenase deficiency hemizygotes.

To study the mechanism of haemolysis in G6PD deficient erythrocytes, studies were undertaken in G6PD deficiency and in normal erythrocytes artificially loaded with calcium. Significantly increased concentrations of calcium, calcium activated neutral protease (CANP) and calcium ATPase were found in patients of G6PD deficiency. However, the membrane bound calcium, the total glycoprotein and sulphydryl groups of membrane were observed to be decreased. Similar results were also observed in the normal erythrocytes when loaded with calcium. These results point to the role of the proteolytic process in membrane modification, and altered membrane permeability during the haemolytic process. Our observations in G6PD deficiency and in in vitro point to the existence of a calcium dependent proteolytic preconditioning of erythrocyte accelerating the haemolysis.

Effect of Ep475 on changes induced by calcium activated neutral protease on erythrocyte membrane.

Increase in cytosolic calcium leads to activation of calcium activated neutral protease (CANP). CANP is known to cause many membrane abnormalities that aid erythrocyte destruction. An epoxy compound Ep475 causes the reversal of such changes induced by calcium and CANP. In absence of Ep475, CANP caused reduction in free sulfhydryl groups and glycoprotein content of the membrane to 61, and 50% respectively, compared to untreated membranes. Calcium ATPase and membrane associated CANP were increased 3 and 1.4 times respectively. Significant reversal of these changes by Ep475 suggests a possible role of this compound in reversing the calcium dependent alterations in RBC including the action of CANP.

Importance of monitoring calcium & calcium related properties in carrier detection for Duchenne muscular dystrophy.

Calcium and calcium dependent enzymes viz., calcium ATPase, protein kinase C and calcium activated neutral protease (milli CANP mCANP) were studied in the erythrocytes, platelets and lymphocytes of obligate carriers, in order to assess the usefulness of these indices for detection of carriers for Duchenne muscular dystrophy (DMD). With the exception of mCANP and lymphocyte calcium ATPase, other calcium dependent enzyme activities showed considerable overlap between carriers and control. Since the increase in the level of platelet mCANP was found in all affected boys (no false negatives) and obligate carriers, and patients with other myopathic conditions and some neurogenic causes did not show high platelet mCANP activity, this parameter could be considered as a good phenotypic index. Unlike SCK, the platelet mCANP of carriers did not overlap that of controls, hence tests are to be carried out to verify its usefulness as an index of carrier state in mutations other than DNA deletion since testing of non-deletion is both costly and has practical limitations.

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation.

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca 2+ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca2+ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.

Human placental calcium activated neutral proteinase: Separation and functional characterization of subunits.

The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.

Calcium activated neutral protease in blood cells from patients & carriers of Duchenne muscular dystrophy.

As blood cells such as platelets, lymphocytes and erythrocytes from patients with Duchenne muscular dystrophy show evidence of membrane alterations and elevation of intra-cellular calcium, one of the calcium related changes i.e., the activity of calcium activated neutral protease (CANP) was monitored and found to be elevated in erythrocytes, lymphocytes and platelets. As similar changes were observed in platelets of carriers of this disease, CANP in platelets may serve as a useful index for carrier detection.