Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 13

FINDING GENES

447

a decade after the first evidence of

linkage, and after extensive characterization

of the

DNA of the region, no specific gene had

emerged as a strong contender for the cause of

the disease. Huntington’s is a case where the disease phenotype is premature death of cer-

tain cells in the brain. Thus genes with expression specific to the brain were plausible

candidates, but many more indirect mechanisms could not be ruled out. Eventually the

gene for Huntington’s was identified because a coding sequence isolated from the region

by exon trapping (Box 13.1) was found to contain a polymorphic (CAG)

n sequence.

However, in less fortunate cases, unless

particular biological hypotheses emerge that can

be tested, one is left with the particularly unpalatable alternative of sequencing the DNA

of the region in a set of affected and unaffected individuals. The disease allele should

be

one that is common to all affected and not common to others. The problem is that in a Mb

region of DNA, there will be several thousand polymorphisms. Only one of these is the

disease allele, itself. The remainder are presumably harmless, normal, silent variations in

the population.

A sensible strategy would focus on

sequencing coding regions first, in the hope

that

quence

are likely to lead to significant

disease phenotypes. Prime

suspects are frame

shifts

that will cause massive changes in protein sequence, and usually premature chain

termination when a stop codon in the new coding region is reached. Insertions, deletions,

and nonconservative amino acid changes are also likely suspects for causing disease. For

example, the protein collagen consists mostly

of a extensive pro-pro-gly

repeat, and the

to

get fooled. For example, many disease alleles in familial Alzheimer’s disease turn out

to

be very conservative amino acid changes like isoleucine to valine. Note that disease al-

human genes, including those responsible for all

forms of inherited disease. Once all

these sequence data are available, the process of

linking specific DNA sequence alter-

ations to particular diseases should become much simpler. In some

cases comparison of

DNA sequence in the human to sequences that have already been studied functionally, in

the human or in other organisms, will provide

direct clues to possible function (see

Chapter 15). In many cases the DNA sequence will allow the construction of useful hy-

bridization probes or PCR primers to examine

the pattern

of gene expression at the

DIAGNOSTICS AT

THE DNA LEVEL

449

There are a number of distinct advantages to the direct examination of DNA for diagnos-

tics. Any cell of the body can be used to detect an inherited alteration in DNA. Thus there

is no need to sample what may be a relatively inaccessible target tissue where the disease

effects will be most pronounced. Since all cells will have the same alteration in DNA,

whether or not this produces a disease phenotype in these particular cells, any cells can be

investigated. In practice, it is most convenient

to

use easily

accessed samples

like

blood

or epithelia sloughed off in saliva.

In principle, diagnosis at the DNA level will be less sensitive than

many

tests

at

the

RNA, protein, or metabolic level. The DNA is present in only a few copies per cell; most

RNAs and proteins or small molecules are much

more frequent, especially

if a cell

in

which the gene or its products are metabolically active is selected. However, for genomic

analysis, it turns out that the sensitivity of DNA testing is more than sufficient. This is es-

pecially true in most cases because PCR amplification can be used, and thus the samples

of cells required can be quite modest in size. The problem is quite different when tests are

applied to tumors or infectious diseases. Here only a small fraction of the target cells may

have altered DNA sequences present. This demands much higher sensitivity, and some-

times DNA tests may not be sensitive enough for the desired analysis, unless potentially

affected cells or infectious agents are purified prior to PCR analysis. However, in some

cases sensitive PCR methods have

been

able

to

detect

low

levels

of

circulating

tumor

cells (Nawroz et al., 1996).

The perfect DNA test will discriminate precisely between all normal alleles in the pop-

ulation and all disease alleles. How close we can come to this ideal is very different for

different inherited diseases. Sickle cell anemia represents an example of the clearest case

for DNA analysis. Here the physical diagnostic criteria are clear enough that a single dis-

ease allele in the population is responsible for the bulk

of

the phenotype we

call

sickle

cell disease. The actual change in the gene of HbA, the normal beta chain of hemoglobin,

to HbS, with a single altered amino acid, occurs in a restriction site for two enzymes. Thus

the disease allele is an RFLP, and it can be analyzed this way to distinguish HbA normals,

HbA HbS carriers (sickle cell trait), and HbS individuals affected with sickle cell disease.

HbA

CCTGAGGAG

Bsu

36 I

CCTNAGG

HbS

CCTGTGGAG

Dde I

CTNAG

Alternatively, allele-specific PCR, as illustrated in Chapter 4, can be used to make the

analysis. There are other hemoglobin disorders, collectively called hemoglobinopathies,

which produce anemia or other impairments in oxygen transport. Many of these can be

successfully analyzed prenatally or in the carrier state by DNA diagnostics. A long cata-

log of specific abnormalities with

particular

clinical presentations

is

now

known. Most

are quite rare.

Cystic fibrosis is an example of a more complex case for DNA diagnostics. This rela-

tively common disease is undoubtedly far more representative of

what will be found for

the majority of human inherited disorders, and it raises a number of severe complications.

In cystic fibrosis, one particular disease allele, a three-base deletion,

resulting

in the

deletion of a single, key amino acid in

the protein chain,

phenylalanine 508,

accounts

for most of the observed disease

cases.

In

populations

of

northern

European extrac-

tion, this allele occurs in about 70% of all affected individuals. (Note the implicit compli-

cation caused by the heterogeneity of

the partially

outbred

human population. The

actual

alleles account for about 20% more of the disease state. Hundreds of additional alleles

must be considered, however, to account for all cystic fibrosis cases that have been exam-

ined thus far at the DNA level. Ironically some of these produce disease that is phenotypi-

cally so different from a classical cystic fibrosis presentation that until DNA analysis

of

this gene defect became prevalent, these cases were not even recognized as being cystic

fibrosis at all. We can extrapolate and conclude that for a large gene like cystic fibrosis,

the number of potential disease alleles in our population will be so large that it may never

be possible to identify all of them a priori. This will certainly be the case if some are

the

likely their

offspring will be unaffected by cystic fibrosis. A negative result in a test for

the next ten

most likely alleles would certainly all but rule out the risk of a child with cys-

ent, one may really want to scrutinize the

second parent for minor cystic fibrosis alleles.

We need to develop much more cost-effective and accurate ways to do this.

The above example illustrates a major

benefit of genetic testing at the DNA level. In a

disease. Thus the family is spared

a great deal of unnecessary anxiety, and the child is

spared the trauma of more invasive

diagnostic tests. A particularly compelling case for

matosis. This is a very unpleasant and disabling disease (the

elephant man syndrome).

The usual earliest clinical presentation of the disease is the

presence, at birth, of occa-

sional cafe au lait ( light brown pigmentation) spots. Many less severe syndromes can also

Genetic analysis at the DNA level becomes much more complex for dominant lethal

diseases like neurofibromatosis. The problem, discussed earlier, is that most if not all

of

the alleles responsible for such diseases have to

be new mutations. Unless there is a re-

stricted pattern of mutation hot spots in the gene

responsible for

the disease, one is faced

with the difficult prospect of having to search the entire

gene for all possible

DNA