Discovery and Biological Basis  

In 1910 a Chicago cardiologist James B. Herrick reported on a very unusual case he had examined. The patient was a 20-year-old West Indies student, attending a dental school in Chicago:

"The young man reported that he had been laid up in a hospital [sometime before Herrick's examination] with what he called muscular rhumatism. His illness had begun with malaise, pain in the back, the muscles of the legs and arms. He had a slight fever and was pale. . . . he had suffered from a bilious attack . . . had vomited and had later noted his urine was dark . . . . He was still, he said, somewhat short of breath."

Fig.1, James B. Herrick (1861-1954)

Just prior to coming to Herrick's clinic, the student had complained:

"For the past five weeks he had been coughing. Two days prior to the examination . . . his cough had grown worse and he had had a slight chill, followed by fever . . . he mentioned also that he felt weak and dizzy, had a headache and [runniness] of the nose. . . . He was fairly well developed physically and was bright and intelligent. . ."

Herrick's intern, Ernest E. Irons, worked up the blood test and noted some very unusal charateristics. As Herrick described them:

"Nucleated reds [red blood cells] were numerous, 74 being seen in a count of 200 leukocytes [white blood cells; also note: normal mature redblood cells lack a nucleus]. The shape of the reds was very irregular, but what especially attracted attention was the large number of thin, elongated, sickle-shaped and crescent-shaped forms. These were seen in fresh secimen no matter in what way the blood was spread on the slide. They were not seen in specimen of blood taken from other individuals at the same time and prepared under exactlysimilar conditions. . . . They were surely not artifacts, nor were they any form of parasite." (Herrick,1910: 518-19)

Fig.2,

To the left are graphic images showing the striking difference in shape between normal red blood cells and a sickled cell. The same cell, as on the left, will show the normal, biconcave (concave on both surfaces) shape when the oxygen concentration is high, and then assume the sickle shape on the right when the oxygen concentration is low. The process is sometimes reversible depending on how long the oxygen level becomes and how long it remains at that level.

 

Herrick's 1910 paper described the condition that later became known as Sickle-Cell Anemia, a disease affecting red blood cells and the hemoglobin they contain. For a closer look at Herrick's original paper, including Iron's photomicrograph of normal and sickle-cell erythrocytes, click here


B. Brief Review of the Biology of Red Blood Cells

Red Blood Cells (RBCs, for short, or technically, erythrocytes) are membrane-bound cells produced by stem cells in the bone marrow. They contain mostly the protein hemoglobin (abbreviated hereafter as Hb), whose function is to bind oxygen molecules (O2) taken in through the lungs and to carry them to tissues throughout the body.

General Physiological Function of Hemoglobin

Molecules of hemoglobin are composed of four protein subunits, each of which has an iron atom at the center of a special ring structure, the porphyrin ring (more on that in Part II). The iron atom has a special affinity for molecular oxygen (O2), such that each subunit picks up an oxygen molecule. Since there are four subunits per hemoglobin molecule, when fully saturated any hemoglobin therefore carries four oxygen molecules (or eight oxygen atoms). Saturation occurs in the lungs, and desaturation in the body tissues, as follows:

In lungs: Hb + 4 O2 ----------------> Hb.O8

In Tissues: HB.O8 -----------------> Hb + 4 O2

Normally, oxygenated hemoglobin is written simply as HbO2, where it is understood that all four subunits are occupied by oxygen. The ability of hemoglobin to reversibly bind and release oxygen is one of its most important characteristics, since if either property were predominant, it could not serve as an effective carrier molecule. Hemoglobin binds oxygen when the oxygen pressure is high, and releases it when the oxygen pressure is low. In the tissues hemoglobin picks up a small percentage (25%) of the carbon dioxide released there and transports it back to the lungs where it is released. The remainder of the carbon dioxide is transported to the lungs as H2CO3, or carbonic acid, most of which dissociates into hydrogen ions and bicarbonale: H2CO3 H+ + HCO3-2

Any significant decrease in amount of functional Hb is known as anemia. All forms of anemia have serious physiological effects because of reduced oxygen delivery to and reduced carbon dioxide removal from the tissues. Sickle cell anemia, in particular, creates serious depletion of oxygen through two mechanisms:

  1. Because of molecule changes within the sickled cell, oxygen-carrying capacity of the blood is greatly reduced;
  2. Because of their peculiar shape, greater rigidity, and tendency to stick together, sickle cells clog smaller vessels in the circulatory system -- the arterioles and capillaries in particular --, preventing the blood from delivering oxygen and nutrients, and removing carbon dioxide and wastes from the tissues.

The schematic diagram shows the changes that occur as sickle or normal red cells release oxygen in the microcirculation. The upper panel shows that normal red cells retain their biconcave shape and move through the microcirculation (capillaries) without problem. In contrast, the hemoglobin polymerizes in sickle red cells when they release oxygen, as shown in the lower panel. Joint Center for Sickle Cell and Thalassemic Disorders,Brigham and Women's Hospital, Boston, MA

In the case of sickle cell anemia, there are two general sorts of effects that result from the reduced oxygen-carrying and deliver capacity of the blood:

a. Short-Term: Because of poor oxygen delivery the individual is frequently out of breath and tires easily;
 
b. Long-term: Oxygen deprivation leads to poor tissue development;
 
c. Hemolysis and clogging of arterioles and capillaries in the lungs, kidneys and liver by sickled cells leads to malfunction of many systems and usually death by the age of 30.
 
Did you ever wonder who the West Indian student was and what ever happened to him? For more information click here.

C. The Genetic Basis of Sickle Cell Anemia

Almost as soon as sickle cell anemia became recognized as a disease in its own right, clinicians noted that it seemed to run in families; moreover, most of those families in the U.S. were of African descent. British and French colonial doctors had also found that sickling was much more common in equatorial Africa and parts of the Indian sub-continent (roughly three times the rate according to the first survey reported by Dr. R. Winston Evans, a pathologist at the West African Military Hospital in Gambia) than in Europe or North America (Evans 1943-44). These observations all suggested that sickle cell anemia might be genetically determined. Why it should be so much more prevalent in primarily tropical regions, however, remained a mystery. In 1923 John Huck, an instructor in clinical microscopy at Johns Hopkins University Medical School, was among the first to study sickle cell anemia by pedigree analysis. His pedigree for one of the two families he investigated is shown below:

Fig.3, Family pedigree chart for family of C.T., as presented in C.C. Guthrie & John Huck (1923) © In 1923. In all such pedigrees, circles represent females and squares males; the particular condition, when observed phenotypically in an individual, is indicated either by words or, more frequently, by blackening in the square or circle. Each horizontal line represents a generation, as indicated by the Roman numerals to the left. There are four generations of the C.T. family shown here.

Guthrie and Huck were not able to draw any firm conclusions about mode of inheritance from these data, but they did clearly suggest that the condition might well be genetic. However, other doctors were skeptical.

In-Text Question 1: It might seem self-evident that because some trait appears frequently in a family line that it is genetically-determined. Suggest at least one reason why this is not necessarily true.

In-Text Answer 1

In the early days of studying sickle cell anemia, the means of determining whether a person was affected by the disease or not was based largely on a blood test developed by Victor Emmel of Washington University Medical School in 1917. Blood was placed on a microscope slide under a cover slip and allowed to sit for several hours. If after that period of time some cells were found to sickle, the individual was determined to have the "sickling disease." However, differences in degree to which individuals showed more or less severe clinical symptoms covered an enormous range, suggesting that the disease simply ran the gamut, as do many diseases, from mild to severe. This belief ran counter to a genetic hypothesis, which at the time was based on a simplified view of Mendelian inheritance in which organisms either showed a trait (dominant) or did not show it (recessive), with the categories of phenotypic expression (the actual characteristics the individual displayed) seen as discrete, for example, white/black, red/green, tall/short, sickle/non-sickle cells, etc..

Several kinds of observations began to suggest that there were, in fact, two quite different forms of sickle cell anemia: a severe and a mild form. At the cellular level Emmel had noted that blood samples of some suspected sickle cell patients only showed sickling if allowed to stand for several days instead of the usual few hours.

At the physiological level studies by Irving J. Sherman (as part of an undergraduate genetics project at Johns Hopkins University in the late 1930s) on the rate of sickling in blood of different patients indicated that there were two very different types of response to changes in oxygen (barometric) pressure , as shown in the graph below.

These two forms of the disease became known first as sickle cell anemia and sickle cell trait (later sickle cell disease). Sickle cell anemia was the more severe form of the disease which left patients debilitated and led to early death; sickle cell trait was the milder form that often showed no overt physiological effects, and did not usually shorten the patient's life. Note from the graph that people with sickle cell anemia show a rather continuous, progressive rate of sickling as atmospheric pressure (hence amount of oxygen available) is decreased; people with sickle cell trait, on the other hand, show almost no sickling until the barometric pressure is quite low (50 mm of mercury, almost one-third the normal value). This latter is called a threshold effect: where a quantitative change (gradual, or quantitative decrease in barometric pressure) ultimately produces a qualitative change (virtually all the cells sickle with just a small change in pressure). The qualitative difference between the two conditions is underscored by the fact that in everyday life people with sickle cell trait rarely show many, or any, complications from the disease and are, indeed, not usually distinguishable from people with non-sickle hemoglobin. Threshold effects are very common in the physical and biological world, and you will encounter a number of them throughout the semester. In addition to showing the dramatic difference between sickle cell disease and sickle cell trait, Sherman's data also showed clearly the effects of deoxygenation on rate of sickling. It became abundantly clear by this point that sickling occurred in any patient when oxygen concentration of the blood was lowered, though it was obviously much more severe for people with the disease than those with the trait.

The difference between the two forms of the sickling condition led to the proposal that sickle cell anemia was a genetic disease that affected either some structural components of the red blood cell membrane, or possibly the hemoglobin molecule itself. Family studies like those carried on by Guthrie and Huck in 1923 did suggest, taken overall, that the sickle cell condition might be inherited in a simple Mendelian fashion: that is, it could be accounted for by postulating a single mutant gene that showed incomplete dominance with respect to the normal gene (in cases of incomplete dominance the heterozygote shows an intermediate phenotye between the homozgous dominant and homozygous recessive conditions). Thus, if the gene for normal hemoglobin were represented as HbA and that for mutant sickle hemoglobin were represented as HbS, then the following genotypes would exist in the population:

HbAHbA

Homozygous dominant, normal individual (Hemoglobin molecules consist of 2 alpha and 2 beta chains)

HbAHbS

Heterozygous, individual with sickle cell trait (Half the individual's hemoglobin molecules consist of 2 alpha and 2 beta chains, and half consists of 2 alpha and 2 S-chains)

HbSHbS

Homozygous recessive individual with sickle cell disease (sickle cell anemia) (All their hemoglobin molecules consist of 2 alpha and 2 S-chains)

The genetic hypothesis was a simple and clear way to understand how and under what conditions the two forms of the pathology appeared in families. If two parents were each heterozygotes, then approximately one-quarter (25%) of their offspring would be expected to show the severe form (sickle cell anemia); another 25% would be normal, and 50% would be heterozygous individuals like the parents, showing sickle cell trait when tested by Emmel's blood test (heterozygotes are also referred to as carriers, that is, they are able to transmit the mutant gene though often not showing any effects of that gene themselves). This scheme is shown below in traditional Mendelian distribution:

Family 1: One parent normal, one parent a carrier:

Parents

HbAHbA
x
HbAHbs

Offspring

HbAHbA
x
HbAHbs

50%

50%

Family 2: Both Parents Heterozygous (Carriers):

Parents

HbAHbs
x
HbAHbs

Offspring

HbAHbA
HbAHbs
HbsHbs

25%
50%
25%

In-Text Question 2: What are the phenotypes (Normal, Sickle cell disease, Sickle cell trait) of each of these three classes of genotypes?

(a) HbAHbA

(b) HbAHbs

(c) HbsHbs

In-Text Question 3: Which of the genotypes listed above would be least likely to pass on their genes to the next generation (i.e., have children)?

People with sickle cell anemia, since many die before they reach reprodctive age or are physically debilitated as adults. People with sickle cell anemia are not sterile and some do have offspring, but the percentage is much lower than people with sickle cell disease.


Dd Localization of the Genetic Defect

If sickle cell anemia is indeed a genetically-determined disease, then in which component of the red blood cell does the defect lie? There are three major components of the red blood cell that could be site of the defect: the cell membrane, the cell's internal scaffolding or cytoskeleton (composed mostly of the protein actin and tubulin), and the hemoglobin molecules that are packed inside (red blood cells do not have most of the other complex internal organelles, including a nucleus, characteristics of other cells, hence among other things, red blood cells do not reproduce).

In-Text Question 4: In general, what sort of experimental design would you have to devise in order to determine which component of the cell -- membrane, cyto-skeletal protein, or hemoglobin -- might be the defective element in sickle cell anemia?

In-Text Answer 4

Two simple experiments are able to rule out the membrane and the cytoskeleton as the locus of sickling.

Red blood cell "ghosts" (cells that have been broken open by physical means -- usually by placing the cells in a hyposmotic medium such as distilled water) can be prepared in such a way that they retain their basic bi-concave shape, even though their hemoglobin contents have been spilled out into the surrounding medium. In 1927 E. Vernon Hahn and Elizabeth Gillespie, a surgeon and intern, respectively, at the University of Indiana Medical School in Indianapolis, used "ghosts" to make an important prediction. They reasoned that:

IF . . . sickling is due to defects in the red blood cell membrane, and
 
IF . . . "ghosts" from sickle cell anemic patients were subjected to lowered oxygen tension (pressure)
 
THEN . . . "ghost cells" ought to show the same sickling phenomenon found in whole cells.

Performing this simple experiment Hahn and Gillespie found that "ghosts" did not sickle, even when the oxygen tension was reduced to zero. They were thus able to reject the membrane hypothesis. This left either the cytoskeleton or the hemoglobin molecule as the likely cause of the sickling phenomenon.

Another experiment, carried out much later, eliminated the cytoskeleton as the source of the sickling process. The proteins actin and tubulin, the most common proteins that make up the cytoskeleton, are composed of many subunits, joined together much as is a builder's scaffolding, where many similar pieces are joined together to produce a lattice-like support. Now, it is possible to produce red blood cells that lack cytoskeleton, and then to subject them to low oxygen concentrations. When this is done on cells from people with sickle cell anemia, it is found that sickling occurs just as it does in normal red blood cells. The cytoskeleton can also be ruled out as the source of the sickling defect. This leaves hemoglobin as the most likely culprit. What, then, is the nature of that defect? The answer to this question is the subject of Web Page II.

Further evidence corroborating the idea that sickle cell disease might be caused by defective hemoglobin molecules was supplied by the discovery, as early as 1925, of Thalassemia, or Cooley's Anemia, that was also thought to be due to defective hemoglobin. Not quite as debilitating as sickle cell, Thalassemia was known to be more prevalent in the Middle East and Africa then elsewhere, and like all anemias, produced unusual fatigue and other side effects in its victims.

A QUESTION OF CERTAINTY

British philosopher of science Karl Popper emphasized some forty years ago that from a logical point of view it is more certain to reject an hypothesis than to confirm one. By this he meant that while adding one more piece of evidence in favor of an hypothesis provides support for the hypothesis, it does not establish the hypothesis with logical certainty. For example, consider the hypothesis that "All green apples are sour." Tasting 10, 100 or 1,000 green apples and finding them all to be sour confirms the hypothesis, but since it is impossible to taste every green apple in existence now or in the future, we can never be sure that all green apples are sour. However, if one green apple turns out to be sweet, then that negative result allows us to reject the hypothesis with certainty. That is, it is logically impossible for all green apples to be sour if just one turns out to be sweet. Thus, only a negative result leads only to certainty in rejecting an hypothesis, while a positive result leads to an uncertain (but not logically wrong) confirmation. In the case of Hahn and Gillespie's work, for example, by obtaining a negative conclusion from their prediction, they could reject with certainty the hypothesis that sickling was due to a membrane-based defect.

In real life, of course, there are ways around Popper's certainty argument. Scientists do not always give up a cherished hypothesis even in the face of negative evidence. For example, proponents of the membrane-based defect in sickle cell anemia could argue that the process of disrupting the cell by experimental means altered the membrane structure so that it no longer responded to low oxygen tension. Such a response would not be illogical or unjustified, but it would take further experimental work to establish that rupturing the cell did not significantly alter membrane structure and thus change the conditions under which sickling would take place. This is biology's version of the uncertainty principle (from physics) in which the act of maniupulating the system alters the very conditions that are being investigated.


E. A Little Philosophy

The slowly evolving picture of sickle cell anemia and its cause represents a process called consilience by nineteenth-century philosopher of science William Whewell (1794-1866) in his monumental work, The Philosophy of the Inductive Sciences (1840). By consilience Whewell meant the coming together of two or more lines of evidence leading to the same conclusions, or theoretical interpretation. For example, Newtonian physics was a classic example of consilience, since it brought together under one explanation (the inverse-square law of gravitation) observations on the motion of the planets, the moon, falling bodies on the earth's surface, and the tides. In the later nineteenth century Darwin's theory of evolution by natural selection would also turn out to be a good example of consilience. Darwin's explanatory principle (descent by modification through the mechanism of natural selection) brought together observations from comparative anatomy, paleontology, biogeography, embryology and animal and plant breeding). The power of consilience lies in the fact that since each line of evidence is independently derived, the fact that they can be brought together under a single explanatory scheme gives weight and probability to the whole scheme.

By the 1940s various approaches to understanding sickle cell anemia boasted several lines of evidence:

  1. Pedigree analysis shows that sickle cell anemia runs in families and can be interpreted as inherited in a simple Mendelian fashion;
  2. Microscopic observation of the blood of people with sickle cell trait shows that their cells sickle, too, but only when oxygen or general air pressure reaches a very low level;
  3. Biochemical studies showed that the hemoglobin molecules of people with sickle cell disease differed from normal hemoglobin molecules;

By themselves, none of these lines of evidence would have been as convincing as they were when considered together.

Recently, it has been discovered that several individuals who have been diagnosed as homozygous recessive for sickle cell hemoglobin (Hbs/Hbs) do not show any noticeable effects of sickle cell anemia. In examining these patients it was found that they continue to produce fetal hemoglobin, a form of the protein that is normally synthesized during fetal development, but which is turned off within a few months after birth. Fetal hemoglobin is similar to adult hemoglobin, though it has a greater affinity for oxygen than adult hemoglobin, and is coded by a different gene. The hemoglobin F gene (HbF) does not carry the mutation found in the sickle cell gene. Although the individuals examined still had sickle cell hemoglobin circulating in their blood, the presence of fetal hemoglobin meant that the oxygen tension of the blood remains high, so the red blood cells do not sickle. What this finding illustrates, in addition to a possible therapeutic potential (see Web Page IV of this website, Part B), is that genetic effects are influenced by the individual organism's own genetic background (the other genes it possesses) as well as by environmental input. It is extremely important to recognize that simply having a gene does not necessarily indicate what the pehnotype will be. Today with a great deal of hype in the press about genes determining all sorts of physical as well as mental and emotional illnesses, it is wise to keep in mind that there is no one-to-one correlation between genotype and phenotype.


WEB PAGE I: TEST YOURSELF

1. The function of hemoglobin is to

(a) pick up oxygen in the lungs and deliver it to the tissues
(b) pick up oxygen in the tissues and deliver it to the lungs
(c) serve only as an oxygen reservoir with other molecules in the blood serving as primary oxygen carriers
(d) keep red blood cells expanded so oxygen can diffuse inside and dissolve in the internal liquid medium

 

Tutorial Answer 1


2. Hemoglobin is a _____ (type of molecule) found _____ (location)

(a) protein / freely circulating in the blood
(b) carbohydrate / freely circulating in the blood
(c) protein / in solution within red blood cells
(d) carbohydrate / in solution within red blood cells
(e) protein / only within the lung tissue

Tutorial Answer 2


3. Sickle cell anemia refers to

(a) a person with the severe form of the disease
(b) a person with the mild form of the disease
(c) the overall condition in which cells sickle, whether mild or severe
(d) any genetic disease affecting hemoglobin

Tutorial Answer 3


4. The debilitating phenotypic effects of sickle cell disease are produced by

(a) the fact that sickled red blood cells cannot transport oxygen, thus depriving the tissues of the oxygen they need for normal growth and development
(b) the fact that sickled red blood cells have a shorter lifetime than normal red blood cells and thus reduce the overall red blood cell (and thus hemoglobin) count
(c) the fact that sickled red blood cells clog small blood vessels such as arterioles and capillaries, shutting off circulation to the affected areas
(d) All of the above

Tutorial Answer 4


5. Anemia is

(a) just another name for sickle cell disease
(b) any condition in which oxygen delivery by red blood cells is diminished
(c) any condition in which the tissues receive an inadequate supply of oxygen
(d) any form of general weakness

Tutorial Answer 5


6. Genetically speaking, sickle cell anemia is considered a case of incomplete dominance because

(a) the two homozygotes are different from each other
(b) the homozygous normal and heterozygous sickle cell trait condition are clinically the same
(c) the homozygous normal, the heterozygous sickle cell trait, and the homozygous sickle cell disease conditions are clinically different from each other
(d) neither the normal HbA nor the HbS gene masks the effects of the other

Tutorial Answer 6


7. A carrier of a condition such as sickle cell anemia is characterized by which of the following hereditary patterns:

(a) A carrier will on the average pass on the gene to one-half of their offspring
(b) A carrier will on the average pass on the gene to all of their offspring
(c) A carrier will on average pass on the gene to none of their own offspring, but only to their grandchildren
(d) All of the above, the pattern depends on whether the condition is complete or incomplete dominance

Tutorial Answer 7


8. Consilience is a particularly powerful form of support for a theory or explanation because (choose the answer that is most complete and speaks most directly to consilience as a form of reasoning)

(a) it provides a variety of kinds of data
(b) it provides independently derived forms of evidence that can all be grouped under one explanation
(c) it provides data that can be tested in other situations and confirmed or rejected
(d) all of the above

Tutorial Answer 8

Natural Sciences Learning Center
Washington University - Biology
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