George R. Honig

Phosphorus-31 spectroscopic determinations of the phosphorus metabolite profiles of blood components: Erythrocytes, reticulocytes, and platelets

Abstract The phosphorus-31 nuclear magnetic resonance spectroscopic profiles of intact rabbit erythrocytes, reticulocytes, and human platelets have been obtained and compared, and the observed resonances identified with known phosphorus-containing compounds. The relative levels of low- and high-energy phosphates in the spectra have been correlated with the metabolic states of these blood components. Spectra of perchloric acid extracts of these tissues have been compared to those from the intact states. Two hitherto unknown resonances have been seen in the orthophosphate diester region of the spectra of platelets and reticulocytes, but not in mature erythrocytes. It was also observed that ferricyanide ion was a suitable substrate for maintaining the viability of reticulocytes in the anaerobic NMR system. Platelets stored at 4° had considerably lower metabolic reserves than either fresh platelets or those stored at room temperature, which showed only slightly diminished stores of high-energy phosphates, in accord with their known survival characteristics. It was concluded from the study that 31 P spectroscopy will be a valid analytical probe in the study of intact blood components in any disease which alters their phosphorus-containing metabolites.

Juvenile myelomonocytic leukemia (JMML) with the hematologic phenotype of severe β thalassemia

A 3‐year‐old Filipino‐American child with recurrent fever, splenomegaly, anemia, and thrombocytopenia, was found to have a hemoglobin F level of 76.9%. His reticulocyte count was elevated (4.3%), and erythroblasts were present in his peripheral blood. The childs erythrocytes were microcytic (MCV 66.9 fl) but his serum ferritin level was normal. His bone marrow at initial presentation demonstrated normal cellularity without an increase in blast cells. The disease progressed with worsening anemia, leukocytosis, and thrombocytopenia, with increased blasts in his marrow and the appearance of a mediastinal mass. His liver, spleen, and lymph nodes were found to be infiltrated with myeloblasts, supporting a diagnosis of juvenile myelomonocytic leukemia (JMML). Analysis of the childs Hb F showed a Gγ/Aγ ratio of 2.2, which was within the characteristic range for JMML. A globin synthesis study using blood reticulocytes showed an α/non‐α globin synthesis ratio of 2.24, typical of severe homozygous β thalassemia. Southern blot analysis of blood‐leukocyte DNA from the patient and his parents demonstrated no apparent abnormality in the β‐globin gene promoter or coding regions. The elevated level of Hb F in this child with JMML appeared to be part of an acquired Cooleys anemia‐like hematologic phenotype. Am. J. Hematol. 58:67–71, 1998.

Homozygous sickle cell disease with coexistent hereditary spherocytosis in three siblings.

Three siblings with the combination of hereditary spherocytosis and sickle cell anemia are described. Two of the children demonstrated a striking degree of hemolysis and massive splenomegaly. In response to repeated transfusions of packed red cells their spleens decreased in size and symptoms were relieved. One child had a splenectomy, which was followed by marked improvement of anemia.

Laboratory Identification, Screening, Education, and Counseling for Abnormal Hemoglobins and Thalassemias

With presently available laboratory methodology, virtually all of the clinically significant hemoglobin disorders as well as their heterozygous carrier states can be identified accurately, rapidly, and by relatively inexpensive means. In addition, most of these conditions can now be reliably detected in the newborn and in the fetus in utero. By the application of these diagnostic techniques, affected carriers can be identified and provided accurate information about their genetic risks, and if desired antenatal diagnosis may also be carried out to determine the hemoglobin genotype of a potentially affected fetus. The development of centers that offer these types of genetic services has progressed rapidly since the early 1970’s, and these programs are continuing to assume increasing importance in many areas of the world. As an introduction to a discussion of these issues, the following section briefly reviews the most commonly employed laboratory methods for the identification of these disorders and their carrier states, as well as the rationale for their application.

The Globin Gene Mutations

The known human globin gene mutations now total more than 400, representing a considerably larger number than for any other human protein system. By far the most numerous of these mutations are those resulting from replacements of single nucleotide bases, and most of these mutant genes are expressed by the synthesis of structurally abnormal globin chains having substitutions of one of their amino acid residues. The substantial number of hemoglobin variants that were well characterized as long as 20 years aga permitted the specific base changes of many of the globin gene mutations to be inferred even before the genetic code was fully known (Smith, 1962). After the complete triplet code was determined it could be shown that virtually all of the amino acid substitutions in these mutant globins could be explained by single base changes in affected codons. Although only few of the genes for the abnormal globins have thus far been sequenced, those that have been analyzed have fully corroborated the mutation codon assignments that were predicted from the genetic code. In many of the other forms of globin structural abnormalities, which include deletions and insertions of amino acid residues, the formation of abnormally shortened or elongated globin chains, and frameshift mutations, the underlying mutations could also be predicted reliably from known codon relationships in the genetic code. It was therefore possible to develop a comprehensive classification of the mutations underlying most of the structural globin variants well before the time that methods for the analysis of globin gene structure became available.

The Geographic Distribution of Globin Gene Variation

With relatively few exeeptions, the globin gene mutations that are tabulated in the Appendix occur only rarely, with a majority of these mutations having been found in only one or a very limited number of individuals or families. Some of the other globin gene mutations have been shown to occur with relatively high frequency, but only in individuals within highly isolated populations. On the other hand, the group that includes Hb S, Hb C, Hb E, and the thalassemias includes enormous numbers of people and extends over a very wide geographical area. (For a more detailed account see Bowman, 1983, and Winter, 1985).

The Genetics of the Human Globin Gene Loci: Formal Genetics and Gene Linkage

Various aspects of globin gene segregation and inheritance have been discussed in earlier chapters. In this section the genetics of the hemoglobins and their mutations are considered more systematically, both to define the specific principles of globin gene inheritance, and to formulate a rational basis for genetic counseling of families affected with these disorders.

The Human Hemoglobins

The hemoglobin is the intensely colored pigment which imparts the red color to the blood; hemoglobin is the most abundant blood protein in man, and represents more than 95% of the soluble protein content of the erythrocytes. The primary functional role of the hemoglobin is the transport of oxygen from the alveolar capillaries of the lungs to the body tissues; an associated function is the binding of carbon dioxide and protons by deoxyhemoglobin, thereby serving to buffer the blood on the venous side of the circulation.

The Human Globin Genes

Only slightly more than 40 years have elapsed since publication of the classic study of Avery, MacLeod, and McCarty (1944) which first showed that genetic information is stored in the form of deoxyribonucleic acid (DNA). Subsequent milestones in the advancement of knowledge of how DNA fulfills its role as a genetic carrier have included the elucidation of the helical structure of the DNA molecule and the complementary relationship of its base sequences (Watson and Crick, 1953a); the determination of how information encoded in the polynucleotide sequences of the DNA molecule is ultimately translated into polypeptides (Jacob and Monod, 1961); and the delineation of the genetic code (Nirenberg and Leder, 1964) demonstrating the nature and specificity of messenger RNA nucleotide base sequences.

Approaches to the Treatment of the Hemoglobin Disorders

Many of the hemoglobin disorders, particularly those that include the sickle hemoglobinopathies and thalassemia syndromes, produce severe disease manifestations and early mortality in a large percentage of affected individuals. The disability, pain, and loss of productivity that these individuals suffer, as well as their often extensive needs for transfusions and other forms of medical care, have stimulated major efforts toward the development of more effective forms of therapy for these conditions.