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OCR for page 212
STARCH ELECTROPHORESIS OF HEMOGLOBIN: FINDINGS IN THALASSEMIA SYNDROMES PARK S. GERALD * For the past year we have employed starch electrophoresis in the investiga- tion of hemoglobin from patients with various thalassemia syndromes and from their families. The electrophoretic technique which has been used is a modification of that described by Kunkel.i We have utilized this tool primarily ire the study of families in which the propositi were found to have thalassemia major. The diagnosis was based upon the presence of a severe hemolytic anemia faith marked anisocytosis and poi- kilocytosis, nucleated red cells in the peripheral circulation, and hepato- splenomegaly. When not rendered impossible by frequent transfusion, the presence of any other abnormal hemoglobin was excluded by electrophoresis. We have examined twenty-three parents from twelve such families, all of Mediterranean or European ancestry. All but one of these parents showed microcytosis of the red cells. The single normocytic parent was subsequently eliminated from the study when paternity was excluded on the basis of blood groups. Of the twentv-twn Parents with microcvtosis. all hilt one .~howed an . . . 1 A ~ ~1 - elevation in the Ao fraction. l his single exception was further distinguished bv the presence of an abnormal hemoglobin and consequently will be described as a separate entity. The remaining parents, twenty-one in all, comprise a homogeneous group with the following three features in common micro- cytosis, elevated As content and parentage of a thalassemia major child. The simultaneous presence of microcytosis and elevated As content appears at present to be diagnostic of thalassemia trait. As I shall subsequently demon- strate, microcytosis alone, even though hereditarily transmitted, is not a suffi- . . . client criterion. , ~ . o an, . . The degree of elevation of the A, content in the thalassemic parents is of considerable interest. In figure 1 are depicted our most recent data. The values for the normal adults are the results of single analyses, while those for the thalassemic parents are the means of paired analyses. The use of duplicate analyses in the thalassemic population was necessitated by the increase of standard deviation with increase in As content. With two exceptions the thalassemic adults comprise a homogeneous croup. O O ~ Excluding these two ex- ceptions, the average As content is 4.8~. This is surprisingly close to twice 2.5% the median value for the normal group. The two thalassemic parents with As content between 3 and 4~70 would seem to be different from the homogeneous majority. We have studied in detail one of these individuals. Over a two months period the As level has Public Health Service Research Fellow of the National Heart Institute. 212

OCR for page 212
ELECTROPHORETIC FIN1)INGS IN THALASSEMIA GERALD 213 O Normal Adults an LLI c' tar Lie to to o 00 00 000 0 000 000000 2.0 3.0 40 5.0 A2 CONTENT goof total Hqb) 53 Parents of Thalassemia Major Children ooO FIG. 1.- Distribution of the Hb AS content among normal adults and among parents of thalassemia major children. remained constant within the limits of experimental error. Oxide relative has been found with microcytic red cells. His electrophoretic fractionation has similarly given a value for As in this intermediate range. The possibility thus exists that hermetic factors control the degree of elevation of the A2 level in thalassemia trait. I should now like to discuss briefly the parent with microcytosis and an abnormal hemoglobin which I previously mentioned. The propositus for this family clinically appeared to have typical thalassemia major. Examination of the hemoglobin revealed 80: to be alkali-resistar~t and the A2 component comprised an increased amount of the non-alkali-resistant fraction. No ab- normal hemoglobin could be demonstrated. The child's mother exhibited the microcytosis, anisocytosis and poikilocytosis typical of thalassemia trait. Upon electrophoresis of this woman's hemoglobin at pEI 8.6, an abnormal component faith S-like mobility could be detected Dig. 2~. This abnormal hemoglobin was unusual in that it constituted only 11~ of the total pigment present. We have examined further members of this patient's family and have ob- served four additional instances of the same abnormality. All haste had normal or slightly decreased total hemoglobin levels and identical morphologic changes ir, their erythrocytes. All have had a normal As content and 10-12~ of an S-like hemoglobin. Sickling tests were negative in the three individuals FIG. 2. Starch electrophoretic pat- tern of the hemoglobins in the "Le- pore abnormality" and in sickle cell trait. Electrophoresis of the cyanmet- hemoglobin derivatives at pH 8.6 (ve- ronal buyer, ionic strength 0.05 ) duration of run, 40 hours; tempera- ture, 8 C.; distance from origin to front of Hb A', 11.0 cm.

OCR for page 212
214 PART III. ABNORMAL HEMOGLOBINS tested. The consistent Ending of microcytosis and an abnormal hemoglobin in the previously published hemoglobinopathies. Since we have not uniquely characterized the abnormal component by physico-chemical means, we have refrained from designating it as a new hemoglobin. Instead we are calling the syndrome the "lLepore abnormality" after the family in which it was found. From the f~miIV data available, Aid. 3.), it would appear reasonable to ~ , low concentration distinguish this abnormality from any of conclude that the "Lepore abnormality" is transmitted as a single gene defect. Nonetheless, it is still conceivable that this syndrome, like hemoglobin H disease, may be the result of the interaction of two genes. In particular, the failure to demonstrate the S-like hemoglobin in the propositus could be evi- dence of segregation of such genes. FIG. 3. Family pedi- ~ree depicting the here- ~ ` - 1 1< E; ~ ~ data ry tr ansm~ salon of the "Lepore abnormality." O Normal (~)Thalassem~a trait Lepore abnormality Thalassemia major During the course of our studies on thalassemia, we encountered two patients (one of Filipino and one of Italian ancestry) who appeared to have very mild forms of thalassemia major. Electrophoretic analysis proved them to be instances of hemoglobin H disease. These cases were unusual in that the Hb lI comprised only 6~ of the total pigment. Except for this, the find- ir~gs were identical with those of previously published reports; namely, a mild hemolytic anemia, anisocytosis and poikilocytosis of the red cells, formation of inclusions in the erythrocytes after vital staining with cresyl blue, and spleno- megaly. The abnormal hemoglobin moved faster than Al at pH 8.6, had an isoelectric point below pH 6.5, and was denatured by freezing. In addition to the presence of the abnormal hemoglobin, the electrophoretic patterns were unusual in exhibiting a reduction in the amount of the As frac- tion. Quantitation revealed that in both cases the slow component constituted only 1.0% of the total pigment; i.e., less than half the normal amount. Such low levels have otherwise been observed only in very severe iron-deficiency anemia or with high concentration of alkali-resistant hemoglobin. Neither of these conditions was present in our patients. In view of the current hypothesis that hemoglobin H is found only in com- bination with thalassemia trait,2 we had expected an increase in the As con- tent as evidence of the presence of the thalassemia gene. Investigation of the family of the Italian patients partially explained this discrepancy. In figure 4 we have diagrammed the relationship of the family members contacted to date. On the mother's side are have been unable to detect any abnormalities. On