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Genomes, screening and gene therapy: what a sequence is good for

Sequencing a human genome took thirteen years and about two billion pounds the first time and now takes a day. What that buys is three billion letters — which is not a diagnosis, not a list of genes, and certainly not a person. Turning a sequence into something useful is the work, and so is deciding what should be done with it.

Before this Complementary base pairing · The polymerase chain reaction and gel electrophoresis · Non-coding DNA and the genome · Recessive alleles and carriers

Before you start

Gene therapy changes your DNA, so any children you have will inherit the change. This is the misconception that makes public debate about gene therapy almost impossible to follow, because it collapses two things that law and ethics treat entirely separately. Somatic gene therapy alters body cells — lung epithelium, bone marrow, retina — and does nothing whatever to the cells that become sperm or eggs. Nothing is inherited. Indeed the change is often not even permanent in the patient: if the treated cells are not stem cells, they are replaced within weeks by untreated ones and the treatment has to be repeated. Germ line therapy, which alters a gamete or a zygote, genuinely would be inherited by every descendant. That is exactly why every country that has legislated on it has prohibited it in a pregnancy, and why the two are never discussed as one question.

What you should be able to do

Sequencing a genome, and what you actually get

The genome is the complete set of DNA in a cell, coding and non-coding alike. Sequencing it means determining the order of its bases, and it is worth being clear-eyed about what that gives you and what it does not.

The original method, chain termination sequencing, copies a template in the presence of a few modified nucleotides that stop the polymerase wherever one is incorporated. That gives fragments of every possible length, each ending in a known base; separate them by length, read the terminating base of each in turn, and the sequence falls out. Modern high-throughput sequencing keeps the idea and runs millions of short reads in parallel, assembling them by computer.

The change in cost is the fact to carry. The Human Genome Project ran from 1990 to 2003 and cost in the region of two billion pounds to produce one composite human sequence. A human genome can now be sequenced in about a day for a few hundred pounds. That is what turned sequencing from a project into a tool, and it is why questions about personalised medicine have become answerable.

What a genome project produces is a sequence of bases. It does not produce a list of genes: locating them, and working out which non-coding regions do something, is a separate and much slower job called annotation. Nor does a eukaryotic sequence give you the proteome. In a bacterium it very nearly does, because the genes are continuous and one gene gives one polypeptide; in a human, introns must be identified and removed, alternative splicing lets one gene give several polypeptides, large stretches are regulatory rather than coding, and post-translational modification changes the product again. That contrast is a stated specification point on more than one board.

The uses are real enough. Sequencing pathogens identifies strains and tracks how an outbreak spreads, which is how variants of SARS-CoV-2 were followed almost in real time. Comparing genomes between species gives evidence about evolutionary relationships. Comparing them between individuals identifies the alleles associated with disease and, increasingly, which drug a particular patient will respond to.

Probes and microarrays

Once you know a sequence you can go looking for it, and the tool for that is a DNA probe: a short single strand of DNA, typically twenty to a few hundred bases, complementary to the sequence you are hunting, with a label attached. The label is either a radioactive isotope, detected by exposing photographic film, or a fluorescent tag, detected under ultraviolet light.

The method is hybridisation. Sample DNA is made single-stranded by heating, the labelled probe is added, and wherever the probe meets a complementary sequence it binds by hydrogen bonding between complementary bases. Unbound material is washed away, and the label shows where the probe stuck.

The top half is one probe answering one question. The bottom half is the same chemistry run tens of thousands of times side by side, which is the only difference between a probe and an array.

A DNA microarray is a small plate carrying thousands of different single-stranded probes fixed in a grid, each spot a known sequence at a known position. Labelled DNA from the sample is washed over it, unbound material is rinsed off, and the pattern of spots that fluoresce is read by a scanner. Because the position of each probe is known, the pattern is a direct read-out of which of those thousands of sequences the sample contains.

There are two quite different things an array can be used for, and confusing them is a common error. Wash DNA over it and you are asking which alleles the person carries — a genotype. Make cDNA from the mRNA of a tissue and wash that over it instead, and you are asking which genes that tissue is currently transcribing — a pattern of expression. The second is how the expression profile of a tumour is compared with healthy tissue from the same patient, which in turn is how some cancers are now matched to a drug.

DNA probe
A short single-stranded length of DNA, labelled radioactively or fluorescently, with a base sequence complementary to a target sequence.
Hybridisation
The binding of a probe to a complementary single-stranded target sequence by hydrogen bonds between complementary bases.
DNA microarray
A plate carrying many different DNA probes at known positions, allowing thousands of sequences to be tested for at once.

Screening, counselling and what a result means

Genetic screening is testing an individual for particular alleles. In the United Kingdom every newborn is offered the heel-prick blood spot test in the first week of life, which screens for nine conditions including cystic fibrosis and sickle cell disease, because early treatment changes the outcome in every one of them. Adults may be screened as carriers before starting a family, as an unborn fetus by chorionic villus sampling or amniocentesis, as an embryo during IVF before implantation, or predictively for a condition that has not yet appeared.

Genetic counselling is the conversation around the test. A counsellor explains what the test can and cannot show, works out the risk to the person and to any children, sets out the options, and supports whatever decision follows. The professional standard is that counselling is non-directive: the counsellor does not recommend a course of action, because the decision belongs to the family and the counsellor does not have to live with it.

The hardest part of counselling is explaining what a positive result actually says, and two contrasting examples show why.

A woman who tests positive for a pathogenic BRCA1 variant has a lifetime risk of breast cancer of roughly sixty to seventy per cent, against something like twelve to fifteen per cent for women in general. That is a large increase and it is not a diagnosis. Some women with the variant never develop cancer; most women who develop breast cancer do not carry it. The result changes what she might reasonably do — more frequent screening, risk-reducing surgery, or nothing — and it does not tell her what will happen.

A person who tests positive for the Huntington's disease allele is in a different position. Penetrance is essentially complete, so the result does say that the disease will develop, though not when. The reason many people at risk choose not to be tested is precisely that: there is no treatment that changes the course of the disease, so the information alters the years before onset without altering the disease.

That last point is where the ethical arguments live, and they are worth stating as arguments rather than resolving. On one side, knowing allows planning, informed reproductive decisions and earlier surveillance, and withholding a test treats an adult as unable to handle information about their own body. On the other there is a recognised right not to know, testing one person discloses information about relatives who did not consent, and a predictive result can affect employment and insurance — in the UK the Code on Genetic Testing and Insurance bars insurers from requiring predictive results, with one agreed exception for Huntington's disease on life cover above half a million pounds. Prenatal screening carries a further argument, raised most sharply by disabled people's organisations: that screening for conditions such as Down's syndrome communicates a judgement about the worth of lives lived with them, whatever the intentions behind it.

DNA profiling, and the statistic that goes with it

About 98 per cent of the human genome does not code for polypeptides, and scattered through it are short sequences repeated over and over, one after another. These are variable number tandem repeats. The sequence of the repeated unit is the same in everybody; the number of repeats at any given locus varies enormously between people and is inherited like any other allele, one from each parent.

That variation is the basis of DNA profiling. A sample is taken — blood, semen, saliva, a hair root, a few cells left on a surface. Its DNA is extracted and the chosen loci are amplified by PCR, using primers that bind to the constant sequences flanking each repeat region, so the length of each product depends only on how many repeats that person has. The products are separated by electrophoresis, and the pattern of lengths is the profile. The current UK standard examines sixteen such loci plus a marker that indicates sex.

Two people, one locus, four alleles between them. Notice that the repeats themselves are identical in both — what differs is only how many there are, and that is enough.

Because profiling uses non-coding repeats, a profile tells you almost nothing about the person: not their appearance, not their health, not their ancestry in any detailed sense. That is a deliberate design choice and it is a large part of why profiling is considered acceptable where whole-genome sequencing of suspects would not be.

Now the statistic, which is where careful candidates separate themselves from confident ones. A full profile at sixteen loci gives a probability of the order of one in a billion that an unrelated person chosen at random would match. That number is the probability of a matching profile given that the person is not the source. It is emphatically not the probability that the person is not the source given that the profile matches. Reversing those two is called the prosecutor's fallacy, it has contributed to real miscarriages of justice, and an exam question that quotes a match probability is usually asking whether you can spot it.

Three further cautions belong with any profile. Relatives share more alleles than strangers, so the one-in-a-billion figure does not apply to a brother. Degraded or very small samples give partial profiles at fewer loci, and the match probability weakens by orders of magnitude as loci drop out. And a match establishes that this person's DNA is in that sample; it says nothing about when it got there or how, which is a matter for other evidence entirely.

Reading a profile in a paternity case

At one locus a child has alleles of 8 and 12 repeats. The mother has 8 and 15. Man A has 11 and 12; man B has 9 and 15. Which man could be the father, and how confident should you be on this evidence alone?

Work out what the child must have inherited. A child receives one allele at each locus from each parent. The mother carries 8 and 15, so the child's 8 can have come from her. The child's 12 cannot have come from the mother, because she does not carry it, so it must have come from the biological father.

The father must therefore carry a 12 allele at this locus. Man A has 11 and 12, so he could be the father. Man B has 9 and 15 and carries no 12 at all, so he is excluded — and exclusion, unlike inclusion, is definite.

Confidence in man A is another matter. A 12-repeat allele at one locus is not rare; a substantial fraction of the population might carry one. All this result says is that man A is not excluded. A real paternity test examines fifteen or more loci, and it is the product of the probabilities across all of them that produces a figure worth acting on. One matching locus is consistent with paternity, and consistent is not the same as demonstrated.

Gene therapy, and why one kind is contested

Gene therapy means treating a disease by altering the genetic material of the patient's cells — usually by supplying a working allele where the patient has two faulty ones, sometimes by silencing a harmful allele or by correcting it directly. Getting the DNA into cells requires a vector: most often a modified virus, stripped of the genes that let it cause disease and carrying the therapeutic gene instead, or a liposome, a small sphere of phospholipid that fuses with the cell-surface membrane.

One difference, and everything else follows from it. If the altered cells are body cells the change ends with the patient; if they are the cells that become gametes it does not end at all.

Somatic gene therapy alters body cells. It has produced genuine treatments. Children with severe combined immunodeficiency have been treated by removing their bone marrow stem cells, inserting a working allele, and returning them; Luxturna, licensed in 2017, delivers a working RPE65 allele into the retina and restores useful vision in a form of inherited blindness; Casgevy, approved in the UK in 2023, uses gene editing on a patient's own blood stem cells to treat sickle cell disease and β-thalassaemia. Cystic fibrosis remains the standard teaching example and the standard cautionary one: a working CFTR allele can be delivered to airway epithelium in a liposome or a virus, but epithelial cells are replaced continually, so the treatment has to be repeated and the clinical benefit so far has been small.

Somatic therapy has its own difficulties, and honest answers name them. Delivery is inefficient, the immune system may attack a viral vector, and a vector that inserts at random may land inside another gene: in an early trial for severe combined immunodeficiency several children later developed leukaemia because the vector had integrated beside a proto-oncogene. Cost raises a question of justice by itself, with single-dose treatments priced in the millions.

Germ line gene therapy alters a gamete, or a zygote, or an early embryo, so that every cell of the resulting person — including the cells that will make their gametes — carries the change. It is technically possible; in 2018 He Jiankui announced the birth of twin girls whose embryos he had edited, and was subsequently imprisoned in China for three years. Its use in a pregnancy is prohibited by law in every country that has legislated on the question.

The arguments deserve to be set out properly, because this is a disagreement between serious people rather than between the informed and the ignorant.

The case for is that some conditions cannot be treated any other way. A disease that has already damaged an organ during development cannot be undone by treating cells afterwards, and for a couple who both carry two copies of a recessive allele, no embryo selection is possible because every embryo will be affected. Correcting the allele once removes the disease from that family permanently rather than treating each generation in turn, which is arguably kinder and certainly cheaper.

The case against rests on four points that are usually made together. Consent cannot be obtained from the person who is altered, nor from any of their descendants, and they are the people who bear the risk. Editing is not perfectly precise, and an off-target change would be inherited along with the intended one, spreading a new problem through a family line. There is no agreed line between preventing disease and selecting for traits, and once the technique exists the pressure to move the line is unlikely to be zero. And because the technique would be expensive, an ability to give children biological advantages would follow wealth, which some argue would entrench inequality in a form no policy could later reverse.

A third position, held by several national academies, is that germ line editing is not wrong in principle but is not acceptable now, since the safety data do not exist and the mechanisms of oversight do not either. That position matters for exam answers because it shows the argument is not simply for and against: 'not yet, and here are the conditions' is a distinct and defensible view.

TRY IT — Deciding which kind of therapy a case calls for

A couple are both carriers of an autosomal recessive condition that causes severe and irreversible brain damage during fetal development. They ask whether gene therapy could give them an unaffected child. Explain what somatic gene therapy could and could not do for them, what alternatives exist, and why germ line therapy is treated differently in law.

Check your answer

Somatic gene therapy could do very little here, and the reason is timing rather than technique. The damage happens during fetal development, so by the time a child could be treated the brain has already formed abnormally. Supplying a working allele to body cells afterwards cannot undo structural damage that has already occurred; at best it might slow further deterioration.

The alternative that already exists is selection rather than alteration. Both parents are carriers, so on average one embryo in four is affected and three in four are not. Pre-implantation genetic testing during IVF allows embryos to be tested and an unaffected one to be implanted. That involves no editing at all, and it is legal and regulated in the UK. Prenatal diagnosis by chorionic villus sampling or amniocentesis is the other route, with the decision that follows a positive result left to the parents.

Germ line therapy is treated differently because the change would be present in every cell of the resulting person, including the cells that form their gametes, and so would be inherited by every descendant. That raises questions somatic therapy does not: nobody in any future generation can consent, an off-target edit would be inherited alongside the intended one, and there is no way to withdraw the alteration if it turns out to be harmful. Set against that, supporters point out that embryo selection does not help every couple and that correcting an allele once removes the condition from the family for good. The law in every country that has addressed the question currently prohibits it in a pregnancy, which is a decision about risk and consent rather than a statement that the biology is impossible.

In the exam

Check yourself

A biotechnology company offers a mail-order test which, from a saliva sample, reports on a microarray of 700 000 sites and returns a list of conditions the customer is 'at increased risk of'. Evaluate this service: explain what the technique can genuinely establish, three limitations of the information it provides, and one argument on each side of the question of whether such tests should be sold directly to the public.

Answer

What it can establish is real. Saliva contains cells, so DNA can be extracted and amplified by PCR. Washing the labelled DNA over a microarray whose spots carry known probe sequences shows, by hybridisation, which of those 700 000 variants the customer carries. As a determination of genotype at those sites, the result is likely to be accurate.

The first limitation is that a variant associated with a condition is not a cause of it. Most of the associations reported by these tests come from studies that found a particular variant slightly more often in affected people than in unaffected ones. A relative risk of 1.3 against a background risk of 2 per cent moves the absolute risk to about 2.6 per cent, which is a difference most people would ignore if it were presented that way rather than as 'increased risk'.

The second is coverage of the wrong kind. An array tests the sites that were put on it and no others, so a negative result is not reassurance: a customer may carry a rare pathogenic variant in the very gene being reported on and be told nothing, because that variant was not among the 700 000. Nor does an array detect the insertions, deletions and structural changes that cause a great deal of genetic disease.

The third is that risk depends on far more than genotype — environment, diet, age, smoking and family history all contribute — and no counselling is attached, so the customer receives a probability with nobody to explain what it means or what to do.

For direct sale, the strongest argument is autonomy: the information is about the customer's own body, adults are entitled to it without a doctor's permission, and some people act on it usefully. Against it, the strongest argument is harm from misunderstanding: a result presented without counselling can prompt unnecessary anxiety, unnecessary surgery or false reassurance, it discloses information about relatives who never consented, and the burden of sorting out the confusion falls on health services that had no part in selling the test.

Questions

Written to the command words the boards use. Try them on paper before opening a scheme: the marks go to points made, not to length.

Question 14 marks

Explain why the sequenced genome of a bacterium gives its proteome almost directly, while the sequenced genome of a human does not.

Mark scheme
  1. B1 bacterial genes are continuous, with no introns, so one gene gives one polypeptide and the coding sequence can be read straight off the DNA
  2. B1 human genes contain introns, which have to be identified and removed before the coding sequence is known
  3. B1 alternative splicing lets one human gene give several different polypeptides
  4. B1 large stretches of the human genome are regulatory rather than coding, and post-translational modification changes the polypeptide again after translation

Question 24 marks

Compare somatic gene therapy with germ line gene therapy, referring to which cells are altered, what is inherited, and how each stands in law.

Mark scheme
  1. B1 somatic therapy alters body cells such as bone marrow, retina or airway epithelium, whereas germ line therapy alters a gamete, a zygote or an early embryo
  2. B1 a somatic change is not inherited, because the cells that become sperm or eggs are untouched, whereas a germ line change is present in every cell of the resulting person, including those cells, and passes to every descendant
  3. B1 somatic treatment often has to be repeated, because cells that are not stem cells are replaced within weeks, whereas a germ line change is permanent and cannot be withdrawn from the family line
  4. B1 somatic therapy is licensed for several conditions, whereas germ line therapy in a pregnancy is prohibited by law in every country that has legislated on the question

Question 33 marks

Explain how a labelled DNA probe is used to find out whether a person carries a particular allele.

Mark scheme
  1. B1 the probe is a short single strand of DNA whose base sequence is complementary to the sequence being looked for, carrying a radioactive or a fluorescent label
  2. B1 the sample DNA is made single-stranded by heating and the probe is added, and hybridisation occurs wherever the probe meets a complementary sequence, held by hydrogen bonds between complementary bases
  3. B1 unbound probe is washed away, and the label is then detected — on photographic film, or under ultraviolet light — only where the probe has bound

Question 43 marks

A profile from a crime scene matches a suspect, and the court is told that the probability of such a match in an unrelated person chosen at random is one in a billion. A lawyer says there is therefore a one in a billion chance that the suspect is innocent. Explain why that statement is wrong.

Mark scheme
  1. B1 the quoted figure is the probability of obtaining a matching profile given that the person is not the source of the sample
  2. B1 the lawyer has reversed it into the probability that the person is not the source given that the profile matches, which is a different quantity and is not what the figure measures; reversing the two is the prosecutor's fallacy
  3. B1 the figure also assumes an unrelated person, and relatives share far more alleles than strangers, while a degraded sample gives a partial profile at fewer loci and the match probability weakens by orders of magnitude

Question 52 marks

State what a genome project produces, and state what annotation adds to that product afterwards.

Mark scheme
  1. B1 a sequence of bases for the organism, coding and non-coding alike
  2. B1 annotation locates the genes within that sequence and works out which non-coding regions do something, and it is a separate and slower job done afterwards

Worth remembering

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