Genetics Lecture 1

"There's a lot of it about!"

Scarcely a day seems to pass without some reference to genetics in the newspapers. Not since the furore stirred up by the publication of The Origin of Species has Genetics been so prominent in the headlines. Of all sciences which are likely to make changes in our lives in the near future outside the area of microelectronics, no science seems more likely than genetics to have profound effects. As doctors you should understand how genetics is of direct relevance to the lives of patients and you will be expected to deliver advice to patients worried or excited by newspaper stories (which are sometimes poorly written, more usually poorly edited, or based on overoptimism by the researchers concerned.)

In this series of lectures I will attempt to impart some basic principles of the subject (which have not changed since Mendel was rediscovered at the turn of the century) and to show how the techniques of "the new genetics" have revolutionised the subject.

Genetics in the news:

There are a number of headline catching themes, some of which are discussed below.

Forensic science

• DNA fingerprinting

  In 1985 sequences of DNA were discovered which were present at many sites in the genome and which varied in the numbers of copies present at any one location. Human fingerprints though composed of only a few basic elements, lines whorls and loops, are unique to any individual. In the same way, the pattern of variation of these simple DNA elements is sufficient to mark each human individual uniquely (with the exception of identical twins). The added advantage is that any fragment of tissue from which DNA can be extracted, (and this can be as small as a dried drop of blood, or a single hair root) is enough to identify the human from which it originated. The latest high profile trial in which DNA evidence featured was the O.J.Simpson trial. However, in this case although DNA tests established beyond reasonable doubt the identity of various blood samples, enough questions remained as to how the blood came to be present to allow the defendant to be acquitted. This week we heard in the news of a murderer being convicted because of the DNA evidence obtained from minute bloodstains left on the clothes of his victim - herself a doctor at the Royal Free.

  Because the DNA variation is inherited, following the rules of Gregor Mendel which we will discuss in lecture 2, it is also possible to use DNA fingerprinting to establish the relationships between individuals. One of the earliest such uses was in an immigration case to prove that a boy desiring entry to the UK was indeed the son of his mother who was resident here.

• Facial features

  "Forensic scientists will be able to predict a criminal's facial features from a hair root at the scene of the crime" - recent news story. Should we believe this headline? In fact, since the work is going on here at UCL, and since I and my family are some of the experimental guinea pigs, I feel fairly confident to report that, as so often, the position has been overstated by journalists in search of a good headline. Nevertheless, studies are underway to try to find measurable components of facial shapes which are controlled by the action of single genes. The type of feature which can be examined is sometimes subtle and can only be revealed by computer imaging techniques but can be much more obvious such as the prominent cleft in the chins of actors
  Kirk Douglas and his son, Michael.
  Once such features are found then the genes controlling their appearance can be sought using in the first instance genetic linkage analysis which features in lectures 8 and 9

Agriculture

Working in the Galton Laboratory, the first laboratory set up specifically to study human inheritance, and doing research connected with the inheritance of human genetic disease and with the human genome project, I am as guilty as anyone of neglecting the tremendous importance of genetics to agriculture. You may feel that as tomorrow's doctors you too can safely neglect this area. However, as a figure of authority and with your scientific training you are bound to meet from time to time requests for information such as "Is it safe to eat genetically modified food?". In lecture 5 we will consider the techniques of molecular genetics as applied to both human genetics and to agriculture so that even if you lack the detailed information to answer the above question, at least you will understand what processes were involved in creating a "genetically engineered" strain of plants or animals.

Some of the products which have made it to the supermarket shelves include:

• delayed ripening tomatoes
• freeze resistant strawberries
• fast growing fish

Last year Tommy Archer was acquitted in Borchester Crown Court for destroying a field full of genetically modified oilseed rape. Was he right or wrong to fear this crop? (Apologies to those of you who do not follow radio soap opera!) This year true life followed fiction as Lord Peter Melchett was found not guilty on similar charges.

Normal human variation

Human genetics is concerned with the causes and alleviation of disease. However, an important part of the subject is the study of normal human variation, if only to disprove that there is any such thing as a "true Aryan" genetic type. (That name incidentally only means the descendent of a person who spoke the original Aryan language). There are many traits which are the products of variation in the forms of a single gene present in different individuals. Some examples include:

• "uncombable hair" - wild tangly hair with a triangular cross section and a longitudinal groove
• "wooly hair" - hair which forms tight curls
• ear lobes - attached or unattached
• ability to bend the first joint of the thumb back at a right angle
• ability to curl the tongue into a U section
• teeth present at birth - King Louis XIV of France was born in this way, much to the distress of his wet nurses
• colour blindness - there are several forms but the most common, affecting about one male in twelve, is deuteranopia, the inability to distinguish between red and green. It is caused by a missing gene which codes for one of the visual receptor pigments.

Genes can affect behaviour. In some cases we can begin to understand why because the gene in question is responsible for the production of a neurotransmitter or receptor. Other cases are so bizarre that we cannot even begin to guess what is the underlying cause.

Examples of "behavioural" genes include:

• Tourette syndrome - This inherited condition is very variable in its severity. The symptoms vary from no more than minor patterns of repetitive behaviour, motor ticks, to the most notorious symptom, an uncontrollable involuntary urge to utter swearwords and obscenities.
• "The jumping Frenchmen of Maine" - These curiously named people are the descendents of a French immigrant to America in the 18th century. They have an exaggerated startle reflex and will jump to obey any abruptly uttered command however dangerous or foolish it may be.
• Monoamine oxidase deficiency - Monoamine oxidase is an enzyme which acts to destroy certain neurotransmitters such as dopamine. There is an association between deficiency for this enzyme (because of a gene mutation) and the committal of acts of criminal violence.

Ultimately, because almost all genes code for enzyme products, mutant genes give rise to their effects through the altered action of an enzyme. In some of the cases above we can guess what the responsible enzyme might be but in others we have no idea. In the cases below we have a clear understanding of the biochemical defect. In some cases this has led to either a cure or an alleviation.

examples of biochemical deficiencies:

• Phenylketonuria - is caused by the absence of an enzyme which converts phenylalanine to tyrosine. The result is a build up of phenylalanine in the blood to a level where it causes brain damage in infants. Excess phenylalanine is converted into several metabolites and which are excreted into urine where they cause a mousy smell. Understanding the condition has led to a treatment. Patients must be placed on a diet with very little phenylalanine until they are teenagers. The diet must be started within a few weeks of birth, the earlier the better. Everyone born in the UK since the 1950s has been screened within a day or two of birth to test for the presence of this disease. See lecture 6
• favism - an abnormal sensitivity to beans which cause a haemolytic anaemia in patients. Even to smell the flowers of the bean may be enough to trigger an attack. This disease is commoner in the Mediterranean region and was known to Pythagoras who exhorted his pupils to avoid beans. It is caused by deficiency for the enzyme glucose-6-phosphate dehydrogenase.
• Adenosine deaminase deficiency - causes a complete failure of the immune system. It is one of the few diseases which have been successfully treated by gene therapy. Bone marrow cells from patients have been removed, a working gene has been placed into them and the cells have been replaced restoring the immune system.

Although Online Mendelian Inheritance in Man, OMIM, lists 8649 characteristics for which there is some evidence of straightforward, single gene inheritance, there are many more characteristics which do not conform to this simple pattern because they are the result of the aggregate effects of several variable genes and also of interaction with the environment.

"The gene for ....... has been cloned!"

We often seem to hear this. Much to my relief (after working on the project for ten years!) I was finally able to utter these words myself in 1997 about the gene TSC1 which is responsible for a genetic disease, Tuberous sclerosis. What is meant by this statement? And how will it help patients? We will go into fuller detail in lecture 5. For the moment, suffice it to say that when we have identified a mutant gene by "cloning" it, we can often immediately deduce something about its protein product's structure and make a reasonable guess as to its function (see for example the cystic fibrosis story later on). Also, we are able to look for the mutation(s) which may be present in any family with the immediate benefit of being able to detect whether unaffected members of the family are "carriers" (defined later) for instance or to be able to carry out antenatal (or even preimplantation) testing of embryos.

The Human Genome Project (HGP) is a huge international effort to find out the complete DNA sequence of the human genome. The project does not stop there, the 3 billion nucleotides has to be annotated and the detailed structure of each gene has to be aligned to the genomic sequence. The entire DNA sequence of the human genome is scheduled for completion in 2004 and already useful information is starting to be generated. In the next few years more and more "disease" genes are going to be identified based on HGP data. In 1997, the identification both of genes responsible for many cases of breast cancer and the Tuberous sclerosis gene mentioned above were directly aided by the DNA sequence data of the HGP.

Genetics in the near future

One benefit of identifying a genetic disease gene is the potential to offer "gene therapy" - the replacement of the defective gene with a new, functional copy. This is by no means an easy procedure and as yet there have been few successes. However, in the next ten years we will see big advances in our abilities to treat some of the common genetic diseases such as Duchenne Muscular Dystrophy by gene therapy. Gene therapy is also being considered as an approach to fighting cancers and even HIV infection.

A newt can regrow an amputated limb. It would be convenient if humans could also regenerate damaged or missing tissues and organs. The "cloning" of 'Dolly' the sheep from one single cell of adult breast tissue has brought this exciting possibility one step nearer.

Cell Division

We begin with consideration of the mechanics of gene inheritance. You should already be aware that:

• genes are composed of DNA,
• DNA is present as very long molecules,
• the long DNA molecules are complexed with proteins and coiled and folded into a structure known as a chromosome
• the cell has a fairly simple method of ensuring the proper inheritance of its DNA (and hence genes) when it splits into two daughter cells.

It takes many cell divisions to make a person from a single celled egg. In each of those divisions all the genes must be replicated and passed to each daughter cell. For the remainder of this lecture we will concentrate on the process of cell division mitosis and on the structures known as chromosomes.

The Cell Cycle

Interphase

Mitosis

The act of mitosis can be conveniently divided into four phases.

prophase

• The replicated chromosomes become visible and can be seen to be comprised of two sister chromatids still joined together at the centromere,
• the nuclear membrane breaks down,
• centrioles move to opposite poles
• and the spindle forms between them.

metaphase

• The chromosomes attach to the spindle by their kinetochores (which are part of the centromere structure).
• The chromosomes are now at their most condensed.
• They line up at the equator of the spindle.

anaphase

• The centromeres divide
• the chromosomes are pulled apart to the two poles by contracting spindle fibres.

telophase

• Nuclear membranes reform
• the chromosomes decondense.
• The daughter cells return to interphase.

Chromosomes

Chromosomes serve to manoeuvre DNA through the difficulties of cell division where something like two metres of DNA has to be moved through a distance of only about 100 µm and be separated from a similar amount of DNA moving the other way.

Humans have 23 pairs of chromosomes. Twenty two pairs, the autosomes, are the same in either sex and are numbered from 1 - 22 in order of diminishing size. One pair, the sex chromosomes are either a pair of X chromosomes (in females) or an X and the very much smaller Y chromosome (in males). One complete set of chromosomes i.e. autosomes 1-22 and a sex chromosome is known as a haploid set. Cells which contain two complete sets (i.e. most cells except for mature germ cells) are diploid. Each chromosome contains its own unique sequence of DNA. Consequently, when the chromosomal DNA and its associated histone and non-histone protein is at its most densely packed (i.e. at mitotic metaphase) it adopts a shape which is slightly different from any other chromosome.

Cytogeneticists study chromosomes microscopically. Cells are treated with a drug which prevents the spindle fibres forming. The chromosomes continue to be prepared for mitosis but because the spindle has not formed the division remains blocked and cells accumulate at metaphase. The cells can then be "fixed" i.e. treated with a chemical which denatures proteins causing the structures to be preserved, and then burst open on a microscope slide. After gentle treatment with a very small amount of proteinase, the chromosomes are stained. Each chromosome reveals a characteristic pattern of alternating dark and light bands which reflects in some way its underlying architecture. On the right for example is the ideogram which represents the characteristic banding pattern of chromosome 9.

The "chromosome spread" can be photographed, individual chromosomes cut out and paired and displayed as shown below.

The sum of all the chromosome information is known as a karyotype.

All human chromosomes have two arms, the short arm is referred to as the p arm and the long arm as the q. The position of the primary constriction (another name for the centromere) defines whether the chromosome is metacentric (two substantial arms) or acrocentric (one very tiny arm and one which contains almost all the DNA). The acrocentric chromosomes are numbers 13, 14, 15, 21 and 22. At each end of the chromosome is a telomere, a structure designed to avoid problems with DNA replication right to the end of a linear molecule. Acrocentric chromosome short arms sometimes hardly seem to be attached, they can be linked via a short stalk known as a secondary constriction. The tiny short arm, bobbing about at a distance is known as a satellite.

Recommended reading

Topics

• Introduction to human genetics in medicine,
• chromosomes (the naming of parts) and
• mitosis.

Reading

The following are alternatives, read as many as you want - but at least one!

• Mange and Mange Chapters 1, 2 and 3 up to page 34
• Lewis Chapters 1, 2 (There's more in both these books than in the lecture but what the heck, it's all jolly interesting!)
• Mueller and Young Chapter 1 and Chapter 3 (pp 23 - 30 and p43)
• Jorde et al. Chapter 1 and Chapter 2 up to page 25.
• Thompson McInnes and Willard Chapters 1 and 2 (pp 13 - 23), chapter 3 (pp 31 - 40)

• Connor and Ferguson-Smith Chapter 4

• Molecular Medicine by R.J. Trent (2nd edition, Chapter 8 which would bear rereading after lecture 5

SAQs

1. What would happen to a chromosome with no telomeres?
2. If the DNA content of a haploid human germ cell is 3 picograms, how much DNA is present in a normal diploid cell entering mitotic prophase?