Saturday 26 December 2015

Genetics, Environment & Evolution - Part 3

Genetics, Environment & Evolution - Part 3

Examples of Sickle cell disease and thalassaemia

We have seen Familial Hypercholesterolaemia (FH), a condition characterised by a high blood level of cholesterol. We have seen that it results from a genetic mutation (one of several) that that will be continued though generations of the family. The actual moment of mutation has not been observed (investigative genetics  is new) and it could have been in the families for many generations. We have seen that FH can lead to a survival advantage for an individual and ultimately within the family, demonstrated clearly in the 19th century. However under certain circumstances it can lead to a serious health disadvantage and this has been the case during the 20th century of epidemic of coronary heart disease (CHD). Although this disadvantage might shorten life, the genetic mutation will become widespread if major disability and death occur after the years of reproduction.

Relative risk of death in families with FH

It is likely that survival advantage is the result of a higher expression of defensive LDL-cholesterol in the inflammatory process. The disadvantage during the CHD epidemic is due to the increased amount of cholesterol in the inflammatory reaction causing partial or complete blockage of one or more coronary arteries on the surface of the heart.

A parallel with FH is sickle cell disease (SCD) which has a similar genetic pattern and it also demonstrates an interaction between genes and environment.
Distribution of malaria in Africa

In tropical sub-Saharan African countries, malaria is a common and serious problem. It can lead to chronic illness and early death, often in childhood. Malaria causes more than 600,000 deaths per year, 90% in Africa, 77% in children. Much medical effort is put into finding a solution, but nature has gone some way to finding one.

Malaria parasite

The malaria parasite, Plasmodium, is carried by infected female Anopheles mosquitoes, which can pierce the skin and feed on human blood. In the process the malaria parasite is transmitted from the mosquito saliva into the the human blood. When it enters the blood stream it finds a home in the red blood cells within the circulation. There is also a secondary liver phase but that need not concern us at present. The malaria parasite feeds and multiplies within the red cells, and every three days (usually) the red cells burst, releasing the parasites within the blood stream. This causes a characteristic high fever with rigors. 
Malaria parasite in the salivary gland of a mosquito
When the release is every three days it is called tertian malaria (due to Plasmodium vivax or ovale), and less commonly if every four days quaternary malaria (due to Plasmodium malariae). The rigors are more frequent or perhaps more or less continuous in the much more serious Plasmodium falciparum infection.

Whichever is the variety of Plasmodium, the essential host factor is “normal” haemoglobin and thus normal red blood cells, something that is also necessary for good human health. Mutations have occurred at some stage in the past that result in an abnormal haemoglobin molecule, due to a single amino acid variation in the gene. As a result the red blood cells are abnormal in structure and function. 

Sickle cell disease (SCD) is the best-known and most serious mutation, and it is quite common in African people, where malaria is most common. The abnormal type of haemoglobin is called HbS, the normal adult haemoglobin being HbA and in the foetus HbF. The presence of HbS in the red cells results in the red cells tending to deformity, a “sickle” shape, particularly under circumstances of oxygen deprivation.
Sickle cells in the blood

People with SCD are found to be protected against malaria, and it is now known that Plasmodium parasites cannot readily reproduce within the abnormal sickle-shaped red cells, which tend to rupture prematurely before maturation of the Plasmodium offspring. Because of this, people with SCD have a distinct survival advantage when the prevalence of malaria is high, and this is why SCD is common in such countries.

We have therefore another example of a mutation that will result in a disease state, but nevertheless it gives a survival advantage under the environmental circumstance of a high risk of malaria. In West Africa the prevalence of SCD is about 4% (this can be regarded as quite common compared to other genetic abnormalities). In the USA, where there is no malaria, the prevalence of SCD (which therefore provides no advantage) has fallen to 0.25% among the black population and is thought to be still falling. Those with the mutation are now at a survival disadvantage.

SCD is generally considered to be due to a recessive gene, as two inherited genes are necessary for the full disease. It is clear that the homozygous state (two abnormal genes, one from each parent) gives a serious disease, but on the other hand a high level of protection against malaria. The two abnormal genes mean that only the abnormal HbS can be produced within the red cells.

The heterozygous state (inheritance of an abnormal gene from just one parent) is called Sickle Cell Trait. There is some advantage in protection against malaria and just mild disease. There is production of both HbS and normal HbA. The pattern of inheritance is thus similar to FH, mild disease in the heterozygous state and serious disease when homozygous. The use of terms Sickle Cell Disease - homozygous - and Sickle Cell Trait (SCT) - heterozygous - is valuable without trying to define whether the gene is recessive, partially recessive or dominant.

SCD and SCT give an advantage in Africa. But if an African person with inheritance of SCD comes to live in temperate countries such as the UK or the USA, where there is no malaria, the health and survival advantage is no longer relevant. The disease state, SCD, then becomes of over-riding importance, and it might be severe, disabling, chronic, and ultimately it might be fatal. There is no major illness in the heterozygous trait, just low-grade asymptomatic anaemia. This benefit outweighing the disadvantage in the heterozygous state can be called the "heterozygous advantage".

Similar to SCD is Thalassaemia, another genetically determined abnormality of haemoglobin (they are in general called “haemoglobinopathies”). Thalassaemia gives rise to low-grade anaemia that can be mistaken for iron deficiency. The heterozygous state is referred to as thalassaemia minor, and the homozygous state thalassaemia major.
Distribution of Thalassaemia
Thalasaemia also gives some protection against malaria, but both the protection and the disease are not as powerful as with SCD. Whereas SCD is common in Africa, thalassaemia is common in south Asia, where the threat of serious malaria is much less. It has also emerged as an evolutionary advantage but only in areas with malaria. There is again a heterozygous advantage.

Genetics and environment

The interaction between genetic change (mutation) and environment is fundamental to the progress of evolution. 

There has been a genetic advantage to a white skin in people living distant from the tropics this being Neanderthal inheritance. Vitamin D production by the action of the sun on the skin is greater when the skin is not pigmented, and this is an advantage when sun intensity is low. On the other hand a white skin in the tropics is a distinct disadvantage as lack of pigmentation of the skin fails to protect against the damaging effects of intense solar radiation.

We have seen that mutations can under certain circumstances give a survival advantage, and this is the process of evolution. Mutations creating abnormal haemoglobin and red blood cells can give a survival advantage in places where there is a high risk of malaria, but illness when there there is no malaria. It does not appear that there will be an end to malaria, and so sickle cells genes will continue.

In the case of familial hypercholesterolaemia, FH, which gives an improvement of defensive inflammatory processes, this can represent a  positive contribution to evolution. Under a 20th century  environmental circumstances the mutation resulted in a major disadvantage – during the epidemic of coronary heart disease, CHD. This disadvantage appears to have been only temporary, during the little more than 50 years. It is likely that in the future the advantage of hypercholesterolaemia will continue and the genes will spread within the population. With careful record-keeping and research, we will be able to record the effect of this – evolution in action. This is a great opportunity.

Thursday 17 December 2015

Environment & Evolution: Part 2 - Mechanisms of genetics

Genetics, Environment and Evolution

Part 2:       Mechanisms of genetics

We have already seen: 
Part 1 - Example of Familial Hypercholesterolaemia (FH)

This showed how a mutation led initially to a health and survival advantage in members of a large family, but during the 20th century epidemic of coronary heart disease (CHD) this changed into a major disadvantage. The inheritance pattern was recessive with partial penetration in the heterozygous state. This requires further explanation, an understanding of the genetics of inheritance. 

Uni-parent (asexual) reproduction is effectively cloning, meaning that the offspring, starting as a single cell, will be genetically identical to the parent. The cell divides into two offspring, a process of cell division, mitosis. It appears to be simple process, but inheritance can easily go wrong. It is necessary for the chromosomes (strings of genes) to double, to replicate, before cell division occurs. In unicellular organisms this results in two identical offspring from one parent, diploid meaning each has two sets of chromosomes. 

Figure 1: cell division, uni-parent reproduction

However whenever chromosomes replicate there is the opportunity for an error. If the error is a fault in the sequence of amino-acids on a gene then this will be a mutation. It might cause no apparent harm but if it results in a disadvantage to the organism then cell death will occur and the reproductive line will come to an end.

We are familiar with sexual reproduction, in which there are two parents (there does not appear to be any advantage to having more than two parents). It entails gene interchange and genetic recombination. The parents each have genes that occur in pairs, and the offspring acquire one of the pair from each parent, so that a different pair continues in each of the next generation. 

There is a considerable advantage in having bi-parent reproduction, namely dilution of a genetic abnormality, and it came about very early in the evolutionary story. A remarkable but highly detailed and technical account of this is found in the book The Vital Question, by Nick Lane.

In uni-parent reproduction a genetic mutation that occurs in a “parent” will be passed to all offspring and all offspring are identical to the parent. A mutation would bring the line to a rapid end, unless it were to provide a significant evolutionary advantage and this would be exceptionally rare. 

Figure 2: Uni-parent inheritance

But if there are two parents, then although the mutation would be passed from one parent to each offspring, it is unlikely to have an effect as the corresponding gene from the other parent would be normal. It will provide the necessary function in the offspring. This is characteristic of a recessive gene - an abnormal gene will not usually cause a problem unless the corresponding gene from the other parent is similarly abnormal. The reproductive line would continue.
Figure 3: Inheritance of a recessive gene

If a gene is recessive it is only when both genes of the pair are abnormal, one from each parent, that the health change, usually disease, will occur. This would be the homozygous state (homo- = same) and only one in four of the offspring would be affected. If the offspring inherits just one abnormal gene, there will be no disease but the individual will be a heterozygous unaffected carrier. 

Sometimes things are not quite so straightforward. In some genetic conditions, and familial hypercholesterolaemia (FH) is an example of this, there will be severe disease in the homozygous state, but just a milder form of disease in the heterozygous state. This represents partial penetrance of a recessive gene.

Figure 4: Inheritance of a recessive gene that has partial penetrance

A dominant genetic disease will occur if only one of a pair of genes is abnormal, the heterozygous state (hetero- = different), but the abnormality is sufficient to cause disease, two normal genes being necessary for the disease-free state. One abnormal gene of the pair will cause disease. In inheritance of a dominant abnormal gene half of the offspring will have the disease.
Figure 5: Inheritance of a dominant genetic abnormality 

This is very much more rare than a recessive genetic characteristic, as the genetic disadvantage of a dominant gene usually causes early death in 50% of the offspring, before reproduction can occur. An exception is Huntington's disease, a serious early onset neurological disorder with dementia and onset at about the age of 40 years, when reproduction will already have taken place. Being dominant inheritance, it will affect one in two of the offspring. There is no carrier state as a single abnormal gene will cause disease.

Genes code for proteins, many of which are enzymes. If only one of a pair of genes will code for an adequate amount of a specific functioning protein or enzyme, then a mutation would create a recessive gene and the heterozygous state would have no disease. 

If we are to understand familial hypercholesterolaemia, FH, we must be aware of the recessive gene with partial penetration. In heterozygous FH with one abnormal gene there is moderate elevation of serum cholesterol and an increased risk of age-related CHD death. But in homozygous FH, when both genes of the pair are abnormal, there is much more important genetic abnormality, with a very high cholesterol and a very high risk of early death from CHD. This means that in families with FH all offspring will have an elevated cholesterol, but some higher than others.

But do not forget that this genetic “abnormality” was, before the epidemic of CHD, a significant evolutionary step forward giving an important survival advantage.

Further details of the inheritance of Familial Hyercholesterolaemia and also Sickle Cell Disease will follow in a further Blog Post.

Friday 11 December 2015

Familial Hypercholesterolaemia - genes, environment, evolution

Genetics, Environment and Evolution

Part 1 - Example of Familial Hypercholesterolaemia (FH)

 View of Delft, Johannes Vermeer 1660-61

We have seen familial hypercholesterolaemia (FH) in a large family in the Netherlands. 

The members of the large family affected had a major health advantage relative to the general population of the country during the 19th century. This advantage was lost in the 20th century and the family members had a very high mortality rate during the epidemic of coronary heart disease (CHD). 

The 20th century epidemic of CHD in the UK, displaying CHD deaths only.

I have suggested that the early advantage of FH was due to the high level of expression of cholesterol in the defensive inflammatory process, and this assisted in controlling and clearing infections within the body. In the 19th century infections were clearly the major causes of death, especially in the earlier years of life. Apart from the advantage in the family described, it is known that a high level of cholesterol in the blood is associated with reduced risk of pneumonia, post-operative infections, and the development of AIDS. LDL-cholesterol (the "bad" cholesterol)is known to inhibit toxins produced by Staphylococci. It is not "bad" but it an important part of body defences against infections. High cholesterol is of no disadvantage above the age of 50 years:  "After age 50 years there is no increased overall mortality with either high or low serum cholesterol levels.”) and in women of all ages, and it is of  a positive advantage above the age of 70 years.

But when it comes to the inflammatory process in CHD (perhaps or probably in response to infection) the high expression of cholesterol causes excessive swelling. Swelling is part of the inflammatory process, described in the 1st century AD as the four cardinal signs of rubor, tumor, calor, dolor - redness, swelling, heat, pain. Under certain circumstances swelling can cause major problems that might in themselves be life-threatening. Examples are stridor in throat infections in children, intestinal obstruction in Crohn’s disease, and of course myocardial infarction when there is inflammation in the coronary arteries of the heart.

When there was no CHD , the members of the family with a high cholesterol had an advantage but during the epidemic of CHD they had a major disadvantage. Half the age-standardised mortality rate became twice the age-standardised mortality rate.

How did it come about that members of this family in the Netherlands had a high level of cholesterol synthesis and blood level? The metabolic “abnormality” is genetic and therefore its development and obvious widespread distribution within the large family was the result of a mutation at an indefinite time in the past.

The mutation

Mutations, changes in genetic sequence, are common and they are usually of no significance. Several mutations can result in FH but the technical term for the mutation occurring with family from the Netherlands is the V408M mutation. 

If it going to be of clinical significance, a mutation can be expected to be a disadvantage and result in disease. We know of many genetic diseases, rare as they might be. But there is no reason in theory why a mutation cannot lead to an advantage, and clearly this has been the way in which evolution has progressed during the millennia. 

However evolution is a very slow process, so slow that in Homo sapiens we cannot observe it in action. A bacterium such as E Coli  can have up to 70 generations per day, and so evolution might be observed over a short time-scale (for example the emergence of antibiotic resistance) but in Homo sapiens 70 generations will take about 2,000 years. Observing evolution does not appear to be possible. 

But in the family with the FH mutation the Netherlands we do have a glimpse of evolution in action. The family has been investigated with good and reliable records of ages at death during a period of about 150 years. This has allowed age-related mortality rates to be determined, and then compared to the population of the country, allowing the expression of the standardised mortality rate (SMR). 


The relative mortality rate (SMR) of the family with FH in the Netherlands
 during the epidemic of CHD. This shows all-cause mortality.

The family members had a low SMR in the mid-19th century, as low as 50% for women. A 50% reduction in age-related death rate must be regarded as a remarkable evolutionary advantage. This is borne out by the large size of the family. This was in an era when children and young people had a significant risk of death from infectious disease, and it was in the resistance to this that the evolutionary advantage appeared to lie.

A large family with a considerable health advantage (the health advantage would have led to the late size of the family) would spread the genes through normal marriage and breeding outside the family. The genetic advantage would spread gradually into the general population. But something went wrong. With the onset of the unexpected epidemic of CHD in the early 20th century, what was an advantage became a serious disadvantage. Early deaths, high SMR, could have slowed down the spread of the mutation, but deaths would have occurred almost entirely after the age of reproduction. 

However now that the epidemic of CHD deaths has almost come to an end, the relative disadvantage of FH is diminishing and might by now have reverted to an advantage. Unfortunately we do not know current details as the research study has not been continued. The mutation is likely to be spreading once again, and with this the health advantage. 

We can therefore appreciate the evolutionary advantage of a high level of expression of LDL-cholesterol in the defensive inflammatory process. It gives the advantages of:
in the family studied, up to 50% reduction in age-standardised all-cause mortality rate;

  • improved survival rate above the age of 60 years;
  • reduced risk of post-operative infection complications;
  • reduced risk of pneumonia;
  • reduced risk of AIDS.

Although the risk of death from CHD is higher, now that the epidemic of CHD is virtually at an end, the advantages of a high cholesterol expression will become clear.

This gives a huge research opportunity to observe evolution in action, and hopefully advantage of this will be taken in the future, especially in the present era of cheap and extensive genetic investigation of large numbers of people. There is no reason why the study should not be revived.

Further information will follow shortly in the future Blog Posts:

Part 2 - Mechanisms of Genetics

Part 3 - Examples of Sickle Cell Disease and Thalassaemia

Johannes Vermeer - Girl with the pearl earring 1665-67

Tuesday 1 December 2015

Cholesterol - the conclusion from Framingham

Cholesterol - the conclusion from Framingham

Framingham study
Anderson KM, Castelli WP, Levy D. 
Cholesterol and mortality: 30 years of follow-up from the Framingham study. 
JAMA 1987; 257: 2176-2180.

It had been suggested (and perhaps assumed) in the 1950s that coronary heart disease (CHD) was due to “cholesterol” - the diet-cholesterol-heart hypothesis was born.

However there was academic concern that there should be a rigorous investigation into the suggested relationship between cholesterol in the blood and subsequent survival. This led to the Framingham Heart Study.

Location of Framingham USA
Framingham is a town in Massachusetts, close to Boston, and this town was chosen for the study. All residents received (with their permission)  an annual cardiovascular health check. Blood cholesterol was estimated at the onset of the study. Deaths were noted carefully.

After 30 years the data were analysed and survival graphs were generated. They are shown below.

The conclusion is shown at the top of this Post. It is true to say that hardly anyone knows of this result. During the 30 years gestation period of the study the diet-heart-cholesterol hypothesis had become engrained in society. No-one wanted to publicise the fact that it is not true. The truth has remained hidden.

Figure 1
Figure 1 shows survival curves for men aged 56-65 at the onset of the 30 year study. The different graph lines indicate different blood levels of cholesterol. It is clear that the cholesterol level had no influence on survival. As they were more than 56 years of age at the onset it is not surprising that 90% had died after 30 years.

There is no display of women in this age-group. Other studies demonstrate that it would have shown an improved survival for those with the highest blood cholesterol levels.

Figure 2
Figure 2 illustrates the same process for men who were in their 30s at the onset of the study. Those with the best survival had the lowest cholesterol levels. This has been publicised well, but without the information of the restricted age range.

Figure 3

Figure 3 illustrates the same process for women who were in their 30s at the onset of the study.  The overall survival (mean 88% at 30 years) is much better than for men (78% at 30 years, Figure 2). There is just a very small effect of cholesterol.

The continuing dogma that cholesterol is always bad is not correct.

Friday 30 October 2015

A family with a high cholesterol - revision

                Canal side, Amsterdam, Netherlands

[Apologies for poor formatting in previous post]

Familial hypercholesterolaemia (FH) is a condition that is genetically determined, and it causes a high level of cholesterol in the blood.  The families affected have experienced a high incidence of coronary heart disease (CHD) with early deaths. This has been a serious problem for them during the second half of the 20th century, and as a consequence there has been a great deal of attention given to these families. But the connection between FH and CHD is not entirely straightforward.

The seriously flawed diet-cholesterol-heart hypothesis of the cause of CHD has dominated medical “non-thinking” up to the present time. It has a variant, the genetic-cholesterol-heart hypothesis. It has become clear to most informed people that diet cannot be the cause of CHD. It has also become clear that the great majority of cholesterol in our bodies is synthesised within us and has not been taken in the diet.

CHD, with or without a high cholesterol level in the blood, has run in families very strongly. It has been much more common than would be explained by simple Mendelian inheritance. However FH is genetically determined and it has a pattern of Mendelian inheritance, as we will see.

Gregor Mendel, the father of genetics

A high level of cholesterol in the blood can lead to deposition of cholesterol in body tissues, but this does not necessarily include the coronary arteries on the surface of the heart. We have seen the example of the high blood levels of cholesterol typical of the liver disease primary biliary cirrhosis (PBC) and it does not lead to an increased risk of CHD. It is important to appreciate that CHD is not due to “cholesterol”, and that cholesterol within the arterial wall is a secondary phenomenon, secondary to an underlying inflammatory process.

CHD is not a dietary disease and it is not a metabolic disease. It is an inflammatory disease. This was first recognised by Rudolf Virchow in the late 19th century but then, eclipsed by the diet-cholesterol-heart hypothesis, it remained forgotten until the late 20th century.

Rudolf Virchow, 1821-1902

Although CHD clearly runs strongly in families, this is a form of non-genetic inheritance, probably the inheritance of a micro-organism rather than genes. However there is clearly an important genetic component in familial hypercholesterolaemia (FH). CHD is worse in these families, for reasons that I hope will become clear. Also, that in the long term things are not altogether bad for those people with FH.

The epidemic of CHD

During the 20th century there was an epidemic of CHD and this is beyond dispute, that is by people who have taken the trouble to read the original papers which have described very clearly its emergence and its decline. 

Figure 1: The epidemic of coronary heart disease in the UK

During this epidemic people with FH fared very badly and had earlier deaths than the average population. Their standardised mortality ratio (SMR) reached almost twice that of the general population.

Very full records have been identified for a large family in The Netherlands, including ages at death over a long period of time. It is now known that the members of this family have FH. From the records, mortality rates have been compared with the population average (mean). If we compare an individual with the average then we are looking at his or her standardised mortality ratio (SMR). The average reference level is 100 and an SMR less than 100 indicates a survival advantage. An SMR of more than 100 is what you do not want, a high death rate. It is similar to IQ but with IQ a level greater than 100 is considered to be an advantage. 

The size of the family enabled 250 people to be studied, starting with a single pair of ancestors in the early 19th century. In those tested in more recent years the blood cholesterol levels were measured and were in excess of 10 mmol/L. Even more recently they have been identified as being carriers of the V408M mutation (see next Post). 


The average blood level of cholesterol in the general population is about 5.9 mmol/L, slightly higher in young men with CHD. We can see this in Figure 2. The distribution curve of blood levels of cholesterol is shown, for young men without CHD between the ages of 30 and 50 years. If we then look at the men of the same age with CHD, we see that the curve has shifted slightly to the right, to a slightly higher level. However there is a major degree of overlap.

Figure 2: Distribution of serum cholesterol, men aged 30-50, Framingham study

CHD deaths have been quite rare in this age-group, and have now effectively come to an end. 

In Figure 3 we now look at a slightly older age-group of men, between 50 and 62 years, we see that the distribution curves are identical.

Figure 3: Distribution of serum cholesterol, men aged 50-62, Framingham study

In other words, blood levels of cholesterol are meaningless for the prediction of CHD in this age-group. This has been explained clearly in the original papers from the Framingham study, a 30 year follow up of the town in Massachusetts. The message has not been heard but here it is:

Could the message be any more clear than this?

It obviously has implications for families with high cholesterol, FH.

The family in The Netherlands

We can see the average SMR of the family in the Netherlands in Figure 4. It has changed considerably during the years 1830 to 1999. In the 19th century the family members had very significant survival advantage, with SMR for females of only 45 and for males 70. At that time it was clearly a very good thing to have FH. 

Figure 4: Relative standardised mortality rate (SMR) for a large family with familial hypercholesterolaemia in The Netherlands

The study indicates that mortality of the FH family relative to the general population increased only after 1915. In the mid 20th century the SMR reached a peak of almost 200. This was very serious with premature deaths from CHD in the family members and an SMR twice that of the general population. 

The pattern of death in the families with FH follows that of the epidemic of CHD. The rise of the graph coincides with the emergence of CHD in the 1920s. The peak was reached in about 1960-70 and thereafter the SMR, the relative death rate fell. So when there was a high prevalence of CHD, people with FH had a higher incidence of death from it. As the overall death rate from CHD in the population diminished, the relatively high SMR for people with FH came back to the national average. 

The last point on the graph represents the time period 1970-99. This is the time of a 90% drop in CHD deaths in the UK. 1999 was very different than 1970. It is unfortunate that the study did not divide 1970-99 into two periods of 1970-84 and 1985-99. A large drop of SMR might then have become visible.

It would be fascinating to see how the families with FH have fared since about 1980 when the recordings stopped. I have written to the authors of the paper asking if the investigation of the families will continue but they tell me that this is unlikely to happen. It is a unique opportunity lost. We can only speculate that those with FH will in the 21st century revert to a much better health profile and lower SMR than the overall population.

Genetics and environment

This important study is an excellent example of the interaction between genes and environment. A genetic advantage in one time or place might turn to be a disadvantage in other circumstances. 

Genetic assessment of FH is very important but it becomes more complex. I think it advisable to close this Post here and to publish an additional post very shortly. It will be "FH - Genetics & Mutations".


Mortality over two centuries in large pedigree with familial hypercholesterolaemia: family tree mortality study.
Sijbrands et al.
British Medical Journal 2003; 322: 1019-23.